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IntroductionPeriodontitis is a biofilm-mediated disease that is usually treated by non-surgical biofilm elimination with or without antibiotics. Antibiotic treatment in periodontal patients is typically selected empirically or using qPCR or DNA hybridization methods. These approaches are directed towards establishing the levels of different periodontal pathogens in periodontal pockets to infer the antibiotic treatment. However, current methods are costly and do not consider the antibiotic susceptibility of the whole subgingival biofilm.MethodsIn the current manuscript, we have developed a method to culture subgingival samples ex vivo in a fast, label-free impedance-based system where biofilm growth is monitored in real-time under exposure to different antibiotics, producing results in 4 hours. To test its efficacy, we performed a double-blind, randomized clinical trial where patients were treated with an antibiotic either selected by the hybridization method (n=32) or by the one with the best effect in the ex vivo growth system (n=32).ResultsAntibiotic selection was different in over 80% of the cases. Clinical parameters such as periodontal pocket depth, attachment level, and bleeding upon probing improved in both groups. However, dental plaque was significantly reduced only in the group where antibiotics were selected according to the ex vivo growth. In addition, 16S rRNA sequencing showed a larger reduction in periodontal pathogens and a larger increase in health-associated bacteria in the ex vivo growth group.DiscussionThe results of clinical and microbiological parameters, together with the reduced cost and low analysis time, support the use of the impedance system for improved individualized antibiotic selection.

Candida species cause life-threatening infections with high morbidity and mortality rates and their resistance to conventional therapy is closely linked to biofilm formation. Thus, the development of new approaches to study Candida biofilms and the identification of novel therapeutic strategies could yield improved clinical outcomes. In the current study, we have set up an impedance-based in vitro system to study Candida spp. biofilms in real-time and to evaluate their sensitivity to two conventional antifungal groups used in clinical practice - azoles and echinocandins. Both fluconazole and voriconazole were unable to inhibit biofilm formation in most strains tested, while echinocandins showed biofilm inhibitory capacity at relatively low concentrations (starting from 0.625 mg/L). However, assays performed on 24 h Candida albicans and C. glabrata biofilms revealed that micafungin and caspofungin failed to eradicate mature biofilms at all tested concentrations, evidencing that once formed, Candida spp. biofilms are extremely difficult to eliminate using currently available antifungals. We then evaluated the antifungal and anti-biofilm effect of andrographolide, a natural compound isolated from the plant Andrographis paniculata with known antibiofilm activity on Gram-positive and Gram-negative bacteria. Optical density measures, impedance evaluation, CFU counts, and electron microscopy data showed that andrographolide strongly inhibits planktonic Candida spp. growth and halts Candida spp. biofilm formation in a dose-dependent manner in all tested strains. Moreover, andrographolide was capable of eliminating mature biofilms and viable cell numbers by up to 99.9% in the C. albicans and C. glabrata strains tested, suggesting its potential as a new approach to treat multi-resistant Candida spp. biofilm-related infections.

ABSTRACTCurrent data on the efficacy of antiseptic mouthwashes to reduce viral load are contradictory. Firstly, in vitro data indicate very strong virucidal effects that are not replicated in clinical studies. Secondly, most clinical studies identify a limited effect, do not include a control/placebo group, or do not evaluate viral viability in an infection model. In the current manuscript, we perform a double-blind, randomized clinical trial where salivary viral load was measured before and after the mouthwash, and where saliva samples were also cultured in an in vitro infection model of SARS-CoV-2 to evaluate the effect of mouthwashes on viral viability. Our data show a 90–99% reduction in SARS-CoV-2 salivary copies with one of the tested mouthwashes, although we show that the remaining viruses are mostly viable. In addition, our data suggest that the active ingredient concentration and the overall excipients’ formulation can play an important role; and most importantly, they indicate that the effect is not immediate, being significant at 15 min and having maximum effectiveness after 1 h. Thus, we show that some oral mouthwashes can be useful in reducing viral transmission, although their efficacy must be improved through refined formulations or revised protocols.

ABSTRACTObjective To develop an in vitro model for real-time monitoring of endodontic biofilm growth and evaluate the ex vivo effect of antibiotics on biofilm growth.Material and Methods Root canal samples were taken from 40 patients and inoculated into 96-well plates in a system that measures biofilm growth through electrical impedance. Biofilm bacterial composition at the genus and species level was analyzed by Illumina sequencing. ANCOM-BC corrected data were used to compare bacterial composition after antibiotic treatment through compositional analysis, and to compare microbiological with clinical data.Results The stationary phase was reached at 8 hours. The biofilm formed had a similar bacterial composition to the inoculum, and Enterococcus faecalis was virtually absent from the samples. The bacterial composition and the effect of antibiotics were sample-dependent. Metronidazole was the antibiotic that most inhibited biofilm formation and azithromycin the one that inhibited it in the highest percentage of cases. The antibiotic effect could not be related to the biofilm original bacterial composition.Conclusions The impedance system allowed real-time monitoring of endodontic biofilm formation, and we propose it as a model for ex vivo evaluation of the whole biofilm susceptibility to antimicrobials, as opposed to evaluating antibiotic sensitivity of specific bacterial isolates.

Background: In vitro studies have shown that several oral antiseptics have virucidal activity against SARS-CoV-2. Thus, mouthwashes have been proposed as an easy to implement strategy to reduce viral transmission. However, there are no data measuring SARS-CoV-2 viability after mouthwashes in vivo. Methods: In this randomized double-blind, five-parallel-group, placebo-controlled clinical trial, SARS-CoV-2 salivary viral load (by quantitative PCR) and its infectious capacity (incubating saliva in cell cultures) have been evaluated before and after four different antiseptic mouthwashes and placebo in 54 COVID-19 patients. Results: Contrary to in vitro evidence, salivary viral load was not affected by any of the four tested mouthwashes. Viral culture indicated that cetylpyridinium chloride (CPC) significantly reduced viral infectivity, but only at 1-hour post-mouthwash. Conclusion: These results indicate that some of the mouthwashes currently used to reduce viral infectivity are not efficient in vivo and, furthermore, that this effect is not immediate, generating a false sense of security.Trial registration: ClinicalTrials.gov identifier: NCT04707742..

Biofilm formation and the appearance of persister cells with low metabolic rates are key factors affecting conventional treatment failure and antibiotic resistance. Using impedance-based measurements, crystal violet staining and traditional culture we have studied the biofilm growth dynamics of 13 Pseudomonas aeruginosa strains under the effect of seven conventional antibiotics. Real-time growth quantifications revealed that the exposure of established P. aeruginosa biofilms to certain concentrations of ciprofloxacin, ceftazidime and tobramycin induced the emergence of persister cells, that showed different morphology and pigmentation, as well increased antibiotic resistance. Whole-genome sequencing of wildtype and persister cells identified several SNPs, a genomic inversion and a genomic duplication in one of the strains. However, these mutations were not uniquely associated with persisters, suggesting that the persistent phenotype may be related to metabolic and transcriptional changes. Given that mannitol has been proposed to activate bacterial metabolism, the synergistic combination of mannitol and ciprofloxacin was evaluated on clinical 48 h P. aeruginosa biofilms. When administered at doses ≥320 mg/L, mannitol was capable of preventing persister cell formation by efficiently activating dormant bacteria and making them susceptible to the antibiotic. These results were confirmed using viable colony counting. As the tested ciprofloxacin-mannitol combination appeared to fully eradicate mature biofilms, we conclude that impedance-based biofilm diagnostics, which permits antibiotic susceptibility testing and the identification of persister cells, is of great potential for the clinical practice and could aid in establishing treatment breakpoints for emerging biofilm-related infections.

Streptococcus dentisani 7746, isolated from dental plaque of caries-free individuals, has been shown to have several beneficial effects in vitro which could contribute to promote oral health, including an antimicrobial activity against oral pathogens by the production of bacteriocins and a pH buffering capacity through ammonia production. Previous work has shown that S. dentisani was able to colonize the oral cavity for 2–4 weeks after application. The aim of the present work was to evaluate its clinical efficacy by a randomized, double-blind, placebo-controlled parallel group study. Fifty nine volunteers were enrolled in the study and randomly assigned to a treatment or placebo group. The treatment consisted of a bucco-adhesive gel application (2.5 109 cfu/dose) with a dental splint for 5 min every 48 h, for a period of 1 month (i.e., 14 doses). Dental plaque and saliva samples were collected at baseline, 15 and 30 days after first application, and 15 days after the end of treatment. At baseline, there was a significant correlation between S. dentisani levels and frequency of toothbrushing. Salivary flow, a major factor influencing oral health, was significantly higher in the probiotic group at day 15 compared with the placebo (4.4 and 3.4 ml/5 min, respectively). In the probiotic group, there was a decrease in the amount of dental plaque and in gingival inflammation, but no differences were observed in the placebo group. The probiotic group showed a significant increase in the levels of salivary ammonia and calcium. Finally, Illumina sequencing of plaque samples showed a beneficial shift in bacterial composition at day 30 relative to baseline, with a reduction of several cariogenic organisms and the key players in plaque formation, probably as a result of bacteriocins production. Only 58% of the participants in the probiotic group showed increased plaque levels of S. dentisani at day 30 and 71% by day 45, indicating that the benefits of S. dentisani application could be augmented by improving colonization efficiency. In conclusion, the application of S. dentisani 7746 improved several clinical and microbiological parameters associated with oral health, supporting its use as a probiotic to prevent tooth decay.

Microorganisms grown in biofilms are more resistant to antimicrobial treatment and immune system attacks compared to their planktonic forms. In fact, infections caused by biofilm-forming Staphylococcus aureus and Staphylococcus epidermidis are a large threat for public health, including patients with medical devices. The aim of the current manuscript was to test the effect of dalbavancin, a recently developed lipoglycopeptide antibiotic, alone or in combination with compounds contributing to bacterial cell disaggregation, on staphylococcal biofilm formation and elimination. We used real-time impedance measurements in microtiter plates to study biofilm growth dynamics of S. aureus and S. epidermidis strains, in the absence or presence of dalbavancin, linezolid, vancomycin, cloxacillin, and rifampicin. Further experiments were undertaken to check whether biofilm-detaching compounds such as N-acetylcysteine (NAC) and ficin could enhance dalbavancin efficiency. Real-time dose–response experiments showed that dalbavancin is a highly effective antimicrobial, preventing staphylococcal biofilm formation at low concentrations. Minimum biofilm inhibitory concentrations were up to 22 higher compared to standard E-test values. Dalbavancin was the only antimicrobial that could halt new biofilm formation on established biofilms compared to the other four antibiotics. The addition of NAC decreased dalbavancin efficacy while the combination of dalbavancin with ficin was more efficient than antibiotic alone in preventing growth once the biofilm was established. Results were confirmed by classical biofilm quantification methods such as crystal violet (CV) staining and viable colony counting. Thus, our data support the use of dalbavancin as a promising antimicrobial to treat biofilm-related infections. Our data also highlight that synergistic and antagonistic effects between antibiotics and biofilm-detaching compounds should be carefully tested in order to achieve an efficient treatment that could prevent both biofilm formation and disruption.

Abstract Most public health measures to contain the COVID-19 pandemic are based on preventing the pathogen spread, and the use of oral antiseptics has been proposed as a strategy to reduce transmission risk. The aim of this manuscript is to test the efficacy of mouthwashes to reduce salivary viral load in vivo. This is a multi-centre, blinded, parallel-group, placebo-controlled randomised clinical trial that tests the effect of four mouthwashes (cetylpyridinium chloride, chlorhexidine, povidone-iodine and hydrogen peroxide) in SARS-CoV-2 salivary load measured by qPCR at baseline and 30, 60 and 120 min after the mouthrinse. A fifth group of patients used distilled water mouthrinse as a control. Eighty-four participants were recruited and divided into 12–15 per group. There were no statistically significant changes in salivary viral load after the use of the different mouthwashes. Although oral antiseptics have shown virucidal effects in vitro, our data show that salivary viral load in COVID-19 patients was not affected by the tested treatments. This could reflect that those mouthwashes are not effective in vivo, or that viral particles are not infective but viral RNA is still detected by PCR. Viral infectivity studies after the use of mouthwashes are therefore required. ( https://clinicaltrials.gov/ct2/show/NCT04707742 ; Identifier: NCT04707742)

Background and objectives: We have developed a standardized, easy-to-use in vitro model to study single- and multiple-species oral biofilms in real time through impedance technology, which elucidates the kinetics of biofilm formation in 96-well plates, without the requirement for any further manipulation. Design and Results: Using this system, biofilms of Streptococcus mutans appear to be sugar-dependent and highly resistant to amoxicilin, an antibiotic to which this oral pathogen is highly sensitive in a planktonic state. Saliva, tongue and dental plaque samples were also used as inocula to form multiple-species biofilms. DNA isolation and Illumina sequencing of the biofilms showed that the multi-species biofilms were formed by tens or hundreds of species, had a similar composition to the original inoculum, and included fastidious microorganisms which are important for oral health and disease. As an example of the potential applications of the model, we show that oral biofilms can be inhibited by amoxicilin, but in some cases they are induced by the antibiotic, suggesting the existence of responders and non-responders to a given antibiotic. Conclusions: We therefore propose the system as a valid in vitro model to study oral biofilm dynamics, including their susceptibility to antibiotics, antiseptics or anti-adhesive compounds.

Background: Previous studies have suggested the quorum sensing signal AI-2 as a potential target to prevent the biofilm formation by Streptococcus mutans, a pathogen involved in tooth decay. Objective: To obtain inhibition of biofilm formation by S. mutans by extracts obtained from the marine bacterium Tenacibaculum sp. 20J interfering with the AI-2 quorum sensing system. Design: The AI-2 inhibitory activity was tested with the biosensors Vibrio harveyi BB170 and JMH597. S. mutans ATCC25175 biofilm formation was monitored using impedance real-time measurements with the xCELLigence system®, confocal laser microscopy, and the crystal violet quantification method. Results: The addition of the cell extract from Tenacibaculum sp. 20J reduced biofilm formation in S. mutans ATCC25175 by 40–50% compared to the control without significantly affecting growth. A decrease of almost 40% was also observed in S. oralis DSM20627 and S. dentisani 7747 biofilms. Conclusions: The ability of Tenacibaculum sp. 20J to interfere with AI-2 and inhibit biofilm formation in S. mutans was demonstrated. The results indicate that the inhibition of quorum sensing processes may constitute a suitable strategy for inhibiting dental plaque formation, although additional experiments using mixed biofilm models would be required.