Spiky Magnetic Microparticles Synthesized from Microrod-Stabilized Pickering Emulsion.

Tailoring the microstructure of magnetic microparticles is of vital importance for their applications. Spiky magnetic particles, such as those made from sunflower pollens, have shown promise in single cell treatment and biofilm removal. Synthetic methods that can replicate or extend the functionality of such spiky particles would be advantageous for their widespread utilization. In this work, a wet-chemical method is introduced for spiky magnetic particles that are templated from microrod-stabilized Pickering emulsions. The spiky morphology is generated by the upright attachment of silica microrods at the oil-water interface of oil droplets. Spiky magnetic microparticles with control over the length of the spikes are obtained by dispersing hydrophobic magnetic nanoparticles in the oil phase and photopolymerizing the monomer. The spiky morphology dramatically enhances colloidal stability of these particles in high ionic strength solutions and physiologic media such as human saliva and saline-based biofilm suspension. To demonstrate their utility, the spiky magnetic particles are applied for magnetically controlled removal of oral biofilms and retrieval of bacteria for diagnostic sampling. This method expands the toolbox for engineering microparticle morphology and could promote the fabrication of functional magnetic microrobots.

Postbiotics as candidates in biofilm inhibition in food industries.

Food-borne pathogen-related biofilms in food processing environments pose significant risks to human health. To ensure human and environmental safety, natural substances with anti-microbial properties and generally recognized as safe (GRAS) status are the future disinfectants of the food industry. The use of postbiotics in food products is gaining attention due to their many benefits. Postbiotics are soluble substances produced by probiotics or released after their lysis, such as bacteriocins, biosurfactants (BSs), and exopolysaccharides (EPS). Postbiotics have drawn attention because of their clear chemical structure, safety dose parameters, long shelf life, and the content of various signaling molecules, which may have anti-biofilm and antibacterial activities. The main mechanisms of postbiotics to combat biofilm contain suppression of twitching motility, disturbing quorum sensing (QS), and reduction of virulence factors. However, there are obstacles to using these compounds in the food matrix because some factors (temperature and pH) can limit the anti-biofilm impact of postbiotics. Therefore, by using encapsulation or application of these compounds in packaging films, the effect of interfering factors can be eliminated. This review summarizes the concept and safety of postbiotics, focusing on their antibiofilm effect, as well as discussing the encapsulation of postbiotics and their application in packaging films.

In-vitro effects of novel periodontal scalers with a planar ultrasonic piezoelectric transducer on periodontal biofilm removal, dentine surface roughness, and periodontal ligament fibroblasts adhesion.

OBJECTIVES: To compare ultrasonic scaler prototypes based on a planar piezoelectric transducer with different working frequencies featuring a titanium (Ti-20, Ti-28, and Ti-40) or stainless steel (SS-28) instrument, with a commercially available scaler (com-29) in terms of biofilm removal and reformation, dentine surface roughness and adhesion of periodontal fibroblasts. MATERIALS AND METHODS: A periodontal multi-species biofilm was formed on specimens with dentine slices. Thereafter specimens were instrumented with scalers in a periodontal pocket model or left untreated (control). The remaining biofilms were quantified and allowed to reform on instrumented dentine slices. In addition, fibroblasts were seeded for attachment evaluation after 72 h of incubation. Dentine surface roughness was analyzed before and after instrumentation. RESULTS: All tested instruments reduced the colony-forming unit (cfu) counts by about 3 to 4 log10 and the biofilm quantity (each p < 0.01 vs. control), but with no statistically significant difference between the instrumented groups. After 24-hour biofilm reformation, no differences in cfu counts were observed between any groups, but the biofilm quantity was about 50% in all instrumented groups compared to the control. The attachment of fibroblasts on instrumented dentine was significantly higher than on untreated dentine (p < 0.05), with the exception of Ti-20. The dentine surface roughness was not affected by any instrumentation. CONCLUSIONS: The planar piezoelectric scaler prototypes are able to efficiently remove biofilm without dentine surface alterations, regardless of the operating frequency or instrument material. CLINICAL RELEVANCE: Ultrasonic scalers based on a planar piezoelectric transducer might be an alternative to currently available ultrasonic scalers.

Long-term effect of simulated five years professional mechanical biofilm removal on the luting gap of ceramic restorations.

BACKGROUND: Achieving sufficient professional mechanical biofilm removal (PMPR) can be challenging in supportive periodontal therapy (SPT), particularly in patients with prosthetic restorations. This experimental study aimed to simulate five years of SPT with periodic PMPR near the luting gap of ceramic restorations using a rubber cup with polishing paste (RCP), air polishing with two different low-abrasive powders (LAPA-1: glycine powder, LAPA-2: erythritol powder), and non-professional mechanical cleaning (control group) to measure the extent of volume loss in the luting gap after baseline (∆V = Vbaseline-V1-5; in µm3). METHODS: Two operators randomly performed PMPR ten times for thirty seconds on one of four sides of 30 crown replicas fixed with glass-ionomer cement (CGIZ: n = 15) or adhesive bonding (CAB: n = 15). The replicas were separated in a template during PMPR, and afterward, cleaned for five seconds per side with a sonic brush under flowing water. The artificial aging process between two PMPRs simulated a 5-year SPT with two PMPRs per year. Profilometric measurements were performed at baseline and after each second PMPR to obtain the mean change of ∆V. The statistical evaluation of the data was carried out using nonparametric tests with Bonferroni correction applied for multiple tests. RESULTS: Ninety-six out of 120 sides could be included in the analysis. PMPR methods showed a loss of substance in the luting gap with a ∆V (mean(standard deviation)) of -4.35 × 106(4.8 × 106)µm3 versus 8.79 × 104(1.05 × 106)µm3 for control at V5 (p ≤ 0.001). No significant differences of ∆V1-5 values could be identified in the control (p > 0.05), whereat all PMPRs showed a significant increasing loss of substance per simulated year (p ≤ 0.001). Intergroup comparison identified LAPA-1 as having the highest significant loss of substance determined on CAB (∆V: -1.05 × 107 (7,2 × 106) µm3), followed by LAPA-2 on CAB (∆V: -6.29 × 106 (4,24 × 106) µm3), LAPA-1 on CGIZ (∆V: -4.15 × 106 (3,25 × 106) µm3), LAPA-2 on CGIZ (∆V: -3.0 × 106 (2,23 × 106) µm3), RCP on CAB (∆V: -1.86 × 106 (2,23 × 106) µm3) and CGIZ (∆V: -1.2 × 106 (1,31 × 106) µm3; p ≤ 0.001)). CONCLUSIONS: Within study limitations, all PMPRs caused a significantly higher loss of substance in the luting gap versus control without professional intervention, with the highest values in the CAB group for LAPA-1, LAPA-2 and RCP. Similar findings were observed for CGIZ, although the loss values were lower.

Comparison of the efficacy of Er,Cr:YSGG laser on oral biofilm removal from implant surfaces with various application times for the treatment of peri-implantitis defects: ex vivo study.

PURPOSE: The major struggle in peri-implantitis therapy is the availability of successful decontamination of the infected implant surface. The main hypothesis of this study was the Er,Cr: YSGG laser decontamination efficacy investigation on the infected implant surfaces with various peri-implantitis defects. The primary objective of this study was to decide the efficacy of Er,Cr:YSGG laser as a decontamination tool at various peri-implantitis simulating defects. The secondary objective was to compare the efficacy of the Er,Cr: YSGG laser on oral biofilm removal between two protocols the first protocol (4 cycles at 2.5 min) and the second protocol (5 cycles at 5 min) at various peri-implantitis simulating defects. MATERIALS AND METHODS: A total of 3 subjects whose plaque biofilms formed in-vivo on twenty-four tested implants were divided into four tested groups. Two native implants were tested as controls.The in vitro defect model was computer-aided designed and printed into a 3D-printed model with various anulations in peri-implant infrabony defects, which were 15,30,60,and 90 degrees. RESULTS: Both Er, Cr: YSGG decontamination protocols at 50 mJ (1.5 W/30 Hz), 50% air, and 40% water were effective at reducing the total implant surface area/ biofilm ratio (%), but the second protocol had a markedly greater reduction in the duration of application (5 cycles at 5 min) than did the first protocol (4 cycles at 2.5 min). CONCLUSION: The Er, Cr: YSGG laser is an effective decontamination device in various peri-implantitis defects. The second protocol(5 cycles at 5 min) with greater application time and circles is more effective than the first one. The defect angulation influence the decontamination capability in peri-implantitis therapy. CLINICAL RELEVANCE (SCIENTIFIC RATIONALE FOR STUDY): Clinicians anticipate that the exploration of suitable therapeutic modalities for peri-implantitis therapy is limited by the obvious heterogeneity of the available evidence in the literature and need for a pre-clinical theoretical basis setup. The major challenges associated with peri-implantitis therapy include the successful decontamination of the infected implant surface, the absence of any damage to the treated implant surface with adequate surface roughness, and the biocompatibility of the implant surface, which allows osteoblastic cells to grow on the treated surface and is the key for successful re-osseointegration. Therefore, these are the expected empirical triads that need to be respected for successful peri-implantitis therapy. Failure of one of the triads represents a peri-implantitis therapeutic failure. The Er, Cr: YSGG laser is regarded as one of the expected devices for achieving the required triad. TRIAL REGISTRATION: "Efficacy of Er,Cr YSGG Laser in Treatment of Peri-implantitis". CLINICALTRIALS: gov ID NCT05137821. First Posted date: 30 -11-2021.

Isolation, characterization of Enterococcus phages and their application in control of E. faecalis in milk.

AIMS: Isolation and characterization of Enterococcus phages and application of phage cocktail to control E. faecalis in milk. METHODS AND RESULTS: For phage isolations, double layer agar method was used. Host range of the phages were determined by the spot test. Twelve phages with varying host ranges were isolated. Phages PEF1, PEF7b, and PEF9 with different host ranges and lytic activities were selected for phage cocktails. Compared to two-phages cocktails tested, the cocktail containing all the three phages displayed stronger antibacterial and biofilm removal activities. The cocktail treatment reduced viable E. faecalis in biofilm by 6 log within 6 h at both 30°C and 4°C. In milk, the cocktail gradually reduced the viable count of E. faecalis and the count reached below the lower limit of detection at 48 h at 4°C. CONCLUSION: The strong bactericidal and biofilm removal activities of the phage cocktail suggest the potential of this cocktail as a natural biocontrol agent for combating E. faecalis in milk.

Evaluation of the antifungal and antibiofilm activity of postbiotics derived from Lactobacillus spp. on Penicillium expansoum in vitro and in food model.

Food degradation made by mycotoxigenic molds represents a significant challenge too food security. Postbiotics are associated with soluble compounds liberated by living bacterial cells or their construction release after lysis, and these metabolites offer the host biological action and specific physiological benefits. In this work, the postbiotics from tree strains of Lactobacillus spp. (Limosilactobacillus reuteri ATCC 367, Lacticaseibacillus casei431and Levilactobacillus brevisATCC) were lyophilized, filtered, and tested to evaluate the antimicrobial and anti-biofilm activity in vitro and milk against P. expansoum. Also, to assess the antioxidant efficacy and the free radical scavenging possibility of the postbiotic, DPPH, and ABTS + methods were used. Antimicrobial activity and biofilm removal activity of postbiotics depended on the Lactobacillus strains used. The minimum inhibitory concentration (MIC) of the prepared postbiotic was determined to be 70ug/ml. The lowest minimum effective concentration (MEC) of postbiotics were significantly differed, in the food matrix, and a low MEC index (100 mg/ml) was detected for postbiotic of L. brevis. Postbiotics derived from L. brevis showed the highest antimicrobial activity compared to L. casei and L. reuteri. The postbiotic extracted from Lactobacillus strain may have functional properties (potential antimicrobial and anti-biofilm) in vitro and food models.

Optimizing the use of low-frequency ultrasound for bacterial detachment of in vivo biofilms in dental research-a methodological study.

OBJECTIVES: Low-frequency, low-intensity ultrasound is commonly utilized in various dental research fields to remove biofilms from surfaces, but no clear recommendation exists in dental studies so far. Therefore, this study aims to optimize the sonication procedure for the dental field to efficiently detach bacteria while preserving viability. MATERIALS AND METHODS: Initial biofilm was formed in vivo on bovine enamel slabs (n = 6) which were worn by four healthy participants for 4 h and 24 h. The enamel slabs covered with biofilm were then ultrasonicated ex vivo for various time periods (0, 1, 2, 4, 6 min). Colony-forming units were determined for quantification, and bacteria were identified using MALDI-TOF. Scanning electron microscopic images were taken to also examine the efficiency of ultrasonications for different time periods. RESULTS: Ultrasonication for 1 min resulted in the highest bacterial counts, with at least 4.5-fold number compared to the non-sonicated control (p < 0.05). Most bacteria were detached within the first 2 min of sonication, but there were still bacteria detached afterwards, although significantly fewer (p < 0.0001). The highest bacterial diversity was observed after 1 and 2 min of sonication (p < 0.03). Longer sonication periods negatively affected bacterial counts of anaerobes, Gram-negative bacteria, and bacilli. Scanning electron microscopic images demonstrated the ability of ultrasound to desorb microorganisms, as well as revealing cell damage and remaining bacteria. CONCLUSIONS: With the use of low-frequency, low-intensity ultrasound, significantly higher bacterial counts and diversity can be reached. A shorter sonication time of 1 min shows the best results overall. CLINICAL RELEVANCE: This standardization is recommended to study initial oral biofilms aged up to 24 h to maximize the outcome of experiments and lead to better comparability of studies.

Surface-Textured Mixed-Metal-Oxide Nanocrystals as Efficient Catalysts for ROS Production and Biofilm Eradication.

Next-generation catalysts are urgently needed to tackle the global challenge of antimicrobial resistance. Existing antimicrobials cannot function in the complex and stressful chemical conditions found in biofilms, and as a result, they are unable to infiltrate, diffuse into, and eradicate the biofilm and its associated matrix. Here, we introduce mixed-FeCo-oxide-based surface-textured nanostructures (MTex) as highly efficient magneto-catalytic platforms. These systems can produce defensive ROS over a broad pH range and can effectively diffuse into the biofilm and kill the embedded bacteria. Because the nanostructures are magnetic, biofilm debris can be scraped out of the microchannels. The key antifouling efficacy of MTex originates from the unique surface topography that resembles that of a ploughed field. These are captured as stable textured intermediates during the oxidative annealing and solid-state conversion of ß-FeOOH nanocrystals. These nanoscale surfaces will advance progress toward developing a broad array of new enzyme-like properties at the nanobio interface.

Subgingival air polishing with trehalose powder during supportive periodontal therapy: use of a conical shaped tip during a randomized clinical trial.

BACKGROUND: This study investigated clinical parameters using a new air-polishing device compared to sonic scaling for subgingival biofilm removal during supportive periodontal therapy. The aim was to evaluate noninferiority of air-polishing compared to sonic scaling in deeper periodontal pockets with respect to pocket depth (PD). METHODS: In 44 participants, 2 single-rooted teeth [(PD) ≥ 5 mm] were treated using a split-mouth design. While a new air polishing device with a conical shaped tip was used for the experimental group, sonic scaling was performed in the control group. PD, clinical attachment level (CAL), and bleeding on probing (BOP) were recorded at baseline, (T0) after 3 months (T1) and 6 months (T2). Pain perception was rated using a visual analog scale (VAS; 0 = no pain, 100 = maximum pain). RESULTS: PD and CAL decreased significantly for both groups, while no intergroup differences were found (PD [mean, mm] control T0 5.96, T2 4.75; experimental T0 5.96, T2 4.8; intergroup p = 0.998; CAL [mean, mm] control T0 7.38, T2 5.84; experimental T0 7.28, T2 6.34; intergroup p = 0.368). For BOP, no intergroup differences were found from T0 to T2 (reduction control 42.5%; experimental 46.5% p = 0.398). Pain perception was significantly lower for air polishing (VAS [mean, mm] control 28.8, experimental 12.56; p = 0.006). CONCLUSION: None of the two treatment procedures showed inferior clinical effects with regard to PD, CAL and BOP with air polishing being more comfortable to patients. Trial registration The study was registered in an international trial register on August 14/08/2019, before the start of recruitment (German Clinical Trial Register number DRKS00017844).

Long-Term Succession Shows Interspecies Competition of Geobacter in Exoelectrogenic Biofilms.

Geobacter spp. are well-known exoelectrogenic microorganisms that often predominate acetate-fed biofilms in microbial fuel cells (MFCs) and other bioelectrochemical systems (BESs). By using an amplicon sequence variance analysis (at one nucleotide resolution), we observed a succession between two closely related species (98% similarity in 16S RNA), Geobacter sulfurreducens and Geobacter anodireducens, in the long-term studies (20 months) of MFC biofilms. Geobacter spp. predominated in the near-electrode portion of the biofilm, while the outer layer contained an abundance of aerobes, which may have helped to consume oxygen but reduced the relative abundance of Geobacter. Removal of the outer aerobes by norspermidine washing of biofilms revealed a transition from G. sulfurreducens to G. anodireducens. This succession was also found to occur rapidly in co-cultures in BES tests even in the absence of oxygen, suggesting that oxygen was not a critical factor. G. sulfurreducens likely dominated in early biofilms by its relatively larger cell size and production of extracellular polymeric substances (individual advantages), while G. anodireducens later predominated due to greater cell numbers (quantitative advantage). Our findings revealed the interspecies competition in the long-term evolution of Geobacter genus, providing microscopic insights into Geobacter's niche and competitiveness in complex electroactive microbial consortia.

Evaluation of a systematic digitized training program on the effectivity of subgingival instrumentation with curettes and sonic scalers in vitro.

OBJECTIVES: Whereas the key role of subgingival instrumentation in periodontal therapy is well known, the influence of operators' experience/training with different devices on treatment results is yet uncertain. Therefore, we assessed untrained undergraduate students, working on manikins, as to how effectively they learn to use curettes (GRA) and sonic scalers (AIR); hypothesizing that AIR will result in higher relative cleaning efficacy (RCE) than GRA. MATERIAL AND METHODS: Before baseline evaluation (T0), 30 operators (9 males, 21 females) received a 2-h theoretical lesson for both instruments, followed by a 12-week period with a weekly digitized training program for 45 min. During three sessions (T1-T3), the operators had to instrument six equivalent test teeth with GRA and AIR. At T0-T3, treatment time, proportion of removed simulated biofilm (RCE-b), and hard deposits (RCE-d) were measured. RESULTS: At T0, RCE-b was in mean(SD) 64.18(25.74) % for GRA, 62.25(26.69) % for AIR; (p = 0.172) and RCE-d 85.48(12.32) %/ 65.71(15.27) % (p < 0.001). At T3, operators reached highest RCE-b in both groups (GRA/AIR 71.54(23.90) %/71.75(23.05)%; p = 0.864); RCE-d GRA/AIR: 84.68(16.84) %/77.85(13.98) %; p < 0.001). Both groups achieved shorter treatment times after training. At T3, using curettes was faster (GRA/AIR 16.67(3.31) min/19.80(4.52) min; p < 0.001). CONCLUSIONS: After systematic digitized training, untrained operators were able to clean 70% of the root surfaces with curettes and sonic scalers. CLINICAL RELEVANCE: It can be concluded that a systematic digitized and interactive training program in manikin heads is helpful in the training of root surface debridement.

Influence of motivation and a new digitized training program on undergraduate dental students during preclinical scaling training.

BACKGROUND: The current study evaluated whether a new digitized scaling training program (DTP: n = 30; supervisor-student-ratio 1:10) improves the performance of undergraduate dental student during a preclinical course in regard to two different instruments [sonic scalers (AIR) and Gracey curettes (GRA)] compared to a conventional training program (CTP: n = 19; supervisor-student-ratio 1:4). METHODS: All the participants received a two-hour lecture on both instruments, followed by a 12-week period with a weekly training program lasting 45 min (10 sessions); one group was supported by DTP. At the end of the training phase, all the participants performed the subgingival scaling of six equivalent test teeth using GRA and AIR. Treatment time, proportion of removed simulated biofilm (relative cleaning efficacy, RCE-b) and hard deposits (RCE-d) were recorded. By using a pseudonymized questionnaire with a 5-point Likert scale, self-assessment of scaling effort, handling, root surface roughness/destruction and effectiveness were evaluated. In addition, personal data such as age, gender, handedness, regularity of playing computer games/consoles and previous dental/technical or medical education were elevated and correlated with cleaning efficacy. RESULTS: The DTP participants showed higher effectiveness in RCE-b compared to those who used the CTP with GRA (71.54% vs. 67.23%, p = 0.004) and AIR (71.75% vs. 62.63%, p ≤ 0.001), and the DTP students were faster with both instruments (p ≤ 0.001). For RCE-d, there was no significant difference between the DTP and CTP groups (GRA p = 0.471; AIR p = 0.158), whereas DTP showed better RCE-d results with GRA versus AIR (84.68% vs. 77.85%, p < 0.001). According to the questionnaire, no significant differences were detected between the training groups in terms of self-assessment, handling, treatment time, root surface roughness/destruction or effectiveness of the instruments. The CTP group favored AIR compared to GRA regarding the fatigue effect. The CTP and playing computer games/consoles regularly was correlated with lower RCE-b, whereas previous education in medicine/dentistry was correlated with higher RCE-b values. CONCLUSIONS: Within the limitations of the study, the DTP with a reduced supervision effort compared to the CTP resulted in higher effectiveness and lower instrumentation time for removing simulated biofilms.

Copper oxide nanoparticles as an effective anti-biofilm agent against a copper tolerant marine bacterium, Staphylococcus lentus.

Biofilm formation on antifouling coatings is a serious concern in seawater cooling systems and the maritime industry. A prolific biofilm forming strain (Staphylococcus lentus), possessing high tolerance (>1,000 µg ml-1) to dissolved copper ions (Cu++) was isolated from titanium coupons exposed in the coastal waters of Kalpakkam, east coast of India. S. lentus formed increased biofilm (p < 0.05) at 100 µg ml-1 of Cu++ ions, when compared with the untreated control. To combat biofilm formation of this strain, the efficacy of copper oxide nanoparticles synthesized from copper nitrate by varying the concentrations of hexamine and cetyl trimethyl ammonium bromide (CTAB), was investigated. Complete (100%) inhibition of biofilm formation was observed with plain CuO NP (0.5 M hexamine, uncapped) at 1,000 µg ml-1. Capping with CTAB, influenced the morphology and the purity of the synthesized CuO NPs but did not alter their surface charge. Capping reduced metal ion release from CuO NPs and their antibacterial and anti-biofilm property against S. lentus. Overall, uncapped CuO NPs were effective in controlling biofilm formation of S. lentus. Concurrent release of copper ions and contact mediated physical damage by CuO NPs offer a promising approach to tackle metal tolerant biofilm bacteria.

Demonstration of biofilm removal from type 304 stainless steel using pulsed-waveform electropolishing.

This article describes an electrochemical method to remove bacterial biofilm from a stainless steel (SS) surface using a potential pulse/reverse pulse technique. This technique employs a periodic waveform that consists of anodic and cathodic pulses. The pulses can effectively strip a thin layer of metal off the SS surface, along with the adherent biofilm, in a saline solution. Not only can the pulses effectively remove biofilm from the SS surface, but they also regenerate the original mirror-like shiny surface. The importance of this electrochemical biofilm removal method is its wide applicability for any types of biofilms. That is, instead of directly removing the biofilm, it removes a very thin layer of the metal under the biofilm. Thus, the removal process is independent to the nature of the biofilms. Furthermore, this electrochemical biofilm removal method is rapid (less than 30 s of potential pulse time) and does not require hazardous chemicals.

Biofilm characteristics and evaluation of the sanitation procedures of thermophilic Aeribacillus pallidus E334 biofilms.

The ability of Aeribacillus pallidus E334 to produce pellicle and form a biofilm was studied. Optimal biofilm formation occurred at 60 °C, pH 7.5 and 1.5% NaCl. Extra polymeric substances (EPS) were composed of proteins and eDNA (21.4 kb). E334 formed biofilm on many surfaces, but mostly preferred polypropylene and glass. Using CLSM analysis, the network-like structure of the EPS was observed. The A. pallidus biofilm had a novel eDNA content. DNaseI susceptibility (86.8% removal) of eDNA revealed its importance in mature biofilms, but the purified eDNA was resistant to DNaseI, probably due to its extended folding outside the matrix. Among 15 cleaning agents, biofilms could be removed with alkaline protease and sodium dodecyl sulphate (SDS). The removal of cells from polypropylene and biomass on glass was achieved with combined SDS/alkaline protease treatment. Strong A. pallidus biofilms could cause risks for industrial processes and abiotic surfaces must be taken into consideration in terms of sanitation procedures.

Is biofilm removal properly assessed? Comparison of different quantification methods in a 96-well plate system.

Various methods have been reported to quantify total biofilm or different components of biofilm; however, these methods are often confusedly used, leading to discrepancies and misleading results. In this study, different methods for quantification of biofilm, including those for total biomass, total amount of bacterial cells, viable cell number, and amount of extracellular polymeric substances, were systematically compared in microtiter plates. To evaluate which method is suitable for assessment of biofilm removal and for bacterial killing, biofilm samples were treated with various cleaners possessing removing and/or killing capacities. It was found that most of the methods tested in this study in general exhibited high reproducibility and repeatability. Crystal Violet staining was a simple but reliable method for total biomass quantification. Total bacteria cell numbers could be reliably quantified by the fluorescent DNA-binding dye Acridine Orange. Viable cells could be quantified by either an ATP-based assay or a proliferation assay. Both of these viability methods showed a broad detection range and led to precise measurement. For quantification of proteins in the biofilm, staining with fluorescein isothiocyanate was most suitable. Furthermore, it was revealed that a combination of different methods is required to determine if a cleaner kills or removes biofilm.

Targeting biofilm infections in humans using small scale robotics.

The eradication of drug-resistant microbial biofilms remains an unresolved global health challenge. Small-scale robotics are providing innovative therapeutic and diagnostic approaches with high precision and efficacy. These approaches are rapidly moving from proof-of-concept studies to translational biomedical applications using ex vivo, animal, and clinical models. Here, we discuss the fundamental and translational aspects of how microrobots target the infection sites to disrupt the structural and functional traits of biofilms and their antimicrobial resistance mechanisms. We emphasize current approaches of mechanochemical disruption and on-site drug delivery that are supported by in vivo models and preclinical testing, while also highlighting diagnostics potential. We also discuss clinical translation challenges and provide perspectives for development of microrobotics approaches to combat biofilm infections and biofouling in humans.

Accumulation and removal of Streptococcus mutans biofilm on enamel and root surfaces in vitro.

Objective: This study aimed to quantitatively investigate the accumulation of Streptococcus mutans biofilm on enamel and root surfaces and assess the amount of biofilm removal using (1) experimental toothpaste and (2) water, in a closed system of flow chamber. Methods: Eight sound premolars were embedded in epoxy resin and polished with silicon carbide grinding papers to display enamel and root surfaces. To mimic biofilm, cultures of Streptococcus mutans were prepared and grown on the tooth surfaces over night before they were exposed to either 2 liters of Milli Q water or 2 liters of 40% experimental toothpaste in the flow chamber. The amount of biofilm was measured and quantified in Fluorescence microscopy. Mean fluorescence values were recorded and analysed using Microsoft® Excel® (MS Excel 2016). Results: The ability to grow biofilm was equally present at both the enamel and root surfaces. The use of water and 40% experimental toothpaste showed a significant reduction of areas covered with biofilm on both enamel and root dentin in comparison to untreated surfaces (p < 0.01). Significantly more biofilm was removed from enamel compared to root surfaces when treated with either water and toothpaste (p < 0.01). Slightly less biofilm was removed by the use of water compared to toothpaste on both enamel and root dentin surfaces, although the differences were not statistically significant. Conclusion: The results indicate that less biofilm is removed from the root surfaces than enamel by the use of water and 40% experimental toothpaste in flow chamber. Assessing oral biofilm accumulation and monitoring biofilm formation on enamel and root dentin surfaces give oral health professionals important directions that could strenghten the significance of dental caries prevention. Improving older individuals' oral hygiene practices should therefore be considered an important measure to prevent root caries.

Effect of Dentifrice Ingredients on Volume and Vitality of a Simulated Periodontal Multispecies Biofilm.

The aim of this in vitro study was to investigate the effect of different toothpaste ingredients on biofilm volume and vitality in an established non-contact biofilm removal model. A multi-species biofilm comprising Porphyromonas gingivalis, Streptococcus sanguinis, and Fusobacterium nucleatum was grown on protein-coated titanium disks. Six disks per group were exposed to 4 seconds non-contact brushing using a sonic toothbrush. Four groups assessed slurries containing different ingredients, i.e., dexpanthenol (DP), peppermint oil (PO), cocamidopropyl betaine (CB), and sodium hydroxide (NaOH), one positive control group with the slurry of a toothpaste (POS), and a negative control group with physiological saline (NEG). Biofilm volume and vitality were measured using live-dead staining and confocal laser scanning microscopy. Statistical analysis comprised descriptive statistics and inter-group differences. In the test groups, lowest vitality and volume were found for CB (50.2 ± 11.9%) and PO (3.6 × 105 ± 1.8 × 105 µm3), respectively. Significant differences regarding biofilm vitality were found comparing CB and PO (p = 0.033), CB and NEG (p = 0.014), NaOH and NEG (p = 0.033), and POS and NEG (p = 0.037). However, no significant inter-group differences for biofilm volume were observed. These findings suggest that CB as a toothpaste ingredient had a considerable impact on biofilm vitality even in a non-contact brushing setting, while no considerable impact on biofilm volume was found.