Polymicrobial biofilms related to dental implant diseases: unravelling the critical role of extracellular biofilm matrix.

Biofilms are complex tri-dimensional structures that encase microbial cells in an extracellular matrix comprising self-produced polymeric substances. The matrix rich in extracellular polymeric substance (EPS) contributes to the unique features of biofilm lifestyle and structure, enhancing microbial accretion, biofilm virulence, and antimicrobial resistance. The role of the EPS matrix of biofilms growing on biotic surfaces, especially dental surfaces, is largely unravelled. To date, there is a lack of a broad overview of existing literature concerning the relationship between the EPS matrix and the dental implant environment and its role in implant-related infections. Here, we discuss recent advances in the critical role of the EPS matrix on biofilm growth and virulence on the dental implant surface and its effect on the etiopathogenesis and progression of implant-related infections. Similar to other biofilms associated with human diseases/conditions, EPS-enriched biofilms on implant surfaces promote microbial accumulation, microbiological shift, cross-kingdom interaction, antimicrobial resistance, biofilm virulence, and, consequently, peri-implant tissue damage. But intriguingly, the protagonism of EPS role on implant-related infections and the development of matrix-target therapeutic strategies has been neglected. Finally, we highlight the need for more in-depth analyses of polymicrobial interactions within EPS matrix and EPS-targeting technologies' rationale for disrupting the complex biofilm microenvironment with more outstanding translation to implant applications in the near future.

Efficacy of curcumin-mediated antibacterial photodynamic therapy for oral antisepsis: A systematic review and network meta-analysis of randomized clinical trials.

BACKGROUND: The presence of oral microorganisms resistant to traditional treatment is increasing, thus a search for new therapies is needed. In this context, antimicrobial photodynamic therapy (aPDT) is an approach for the treatment of antibiotic resistant andnon resistant microorganisms. Therefore, the aim of the present study was to conduct a systematic review and meta-analysis of randomized clinical trials of aPDT for oral antisepsis against oral polymicrobial biofilms. METHODS: PubMed, Science Direct, Scopus, SciELO, Lilacs, Cochrane Library and Embase databases were searched. In total, five articles were included for qualitative analysis and four articles were used for quantitative analyses. Bias assessment of the eligible articles was made using the RoB 2 criteria. Network meta-analysis was performed using the random-effect model. Subgroup's analysis was also conducted. The groups evaluated were aPDT, exposure to light only and no treatment at all (control group). The quality of evidence was assessed by CINeMA approach. RESULTS: aPDT mediated by curcumin had significant results in the reducing bacterial load (0.31-0.49 log10 UFC/ I2=0%) when compared with the control group. The included articles were classified as low risk of bias, despite biases detected by allocation and blinding. Moreover, quantitative analysis between aPDT and control group and between light and control group were classified with low risk of confidence rating, while the results from aPDT versus light were classified as moderate risk of confidence rating. CONCLUSION: aPDT has significant efficacy for oral antisepsis, however more randomized clinical trials will be needed to validate the present results.

Multiple surface interaction mechanisms direct the anchoring, co-aggregation and formation of dual-species biofilm between Candida albicans and Helicobacter pylori.

Introduction: Polymicrobial biofilms have a significant impact on pathogenesis of infectious microorganisms. Many human diseases are affected by colonization of multi-species communities affecting negatively the treatments and increase the risks for the health. In particular, in the epithelium of the stomach co-existence between C. albicans and H. pylori has been described, which has been associated to a synergistic effect on ulcer pathogenesis. Objective: The objective of this work was to advance in the understanding of surface interaction between H. pylori and C. albicans for the formation of polymicrobial biofilms. Methods: Studies of microbial surfaces both bacterium, yeast and co-cultures of them were carried out by infrared spectroscopy, deconvolution analysis, transmission and scanning electron microscopies, and optic microscopy. Additional methods were used to contrast the results as dynamic light scattering, contact angle, agarose gel electrophoresis and gene amplification. Results: Several surface interaction mechanisms promote the anchoring of H. pylori on C. albicans, cell co-aggregation, and polymicrobial biofilm formation, main identified interactions were: (i) hydrophobic interactions between non-polar peptide chains and lipid structures, characterized by θw among 84.9 ± 1.6 (γ = 22.78 mJ/m2 with 95.3 of dispersive contribution) and 76.6 ± 3.8 (γ = 17.34 mJ/m2, 40.2 of dispersive contribution) for C. albicans and H. pylori, respectively, (ii) hydrogen bonds between surface components of yeast and bacterium (e.g., -S-H⋅⋅⋅NH2- or -S-H⋅⋅⋅O[bond, double bond]CO-) and (iii) thiol-mediated surface interactions identified by displacements to lower wavenumbers (Δv = 5 cm-1). Evidence of internalization and electrostatic interactions were not evidenced. All observations were congruent with the biofilm formation, including the identification of small-size biostructures (i.e., 122-459 nm) associated with extracellular proteins, extracellular DNA, or outer membrane vesicles were observed characteristic of biofilm formation. Conclusion: It is concluded that biofilm is formed by co-aggregation after anchoring of H. pylori on C. albicans. Several surface interactions were associated with the prevalence of H. pylori, the possibility to find C. albicans in the stomach epithelium infected by H. pylori, but also, strength interactions could be interfering in experimental observations associated with bacterial-DNA detection in culture mixtures.

Mucoadhesive controlled-release formulations containing morin for the control of oral biofilms.

This study aimed to evaluate the antimicrobial and anti-biofilm activity of morin on polymicrobial biofilms and its cytotoxicity in controlled-release films and tablets based on gellan gum. Polymicrobial biofilms were formed from saliva for 48 h under an intermittent exposure regime to 1% sucrose and in contact with films or tablets of gellan gum containing 2 mg of morin each. Acidogenicity, bacterial viability, dry weight and insoluble extracellular polysaccharides from biofilms were evaluated. The cytotoxicity of morin was evaluated in oral keratinocytes. Morin released from the systems reduced the viability of all the microbial groups evaluated, as well as the dry weight and insoluble polysaccharide concentration in the matrix and promoted the control of acidogenicity when compared with the control group without the substance. Morin was cytotoxic only at the highest concentration evaluated. In conclusion, morin is an effective agent and shows antimicrobial and anti-biofilm activity against polymicrobial biofilms.

Characterization of polymicrobial biofilms obtained from saliva or carious lesions in dentin.

This study aimed to compare the formation of polymicrobial biofilms using carious dentin or saliva as inoculum for application in in vitro microbiological studies on caries research. For biofilm growth, combined samples of infected dentin or saliva from three donors were used. The biofilms were grown on glass coverslips, under a regimen of intermittent exposure (6 h day-1) to 1% sucrose for 4 days. Total bacterial loads, as well as specific aciduric bacteria and mutans streptococci loads were quantified and correlated with biofilm acidogenicity and susceptibility to chlorhexidine. The data were evaluated using the Student's-t, Mann Whitney and Kruskal-Wallis tests. The two biofilms showed similar microbial loads (total bacteria, aciduric bacteria and mutans streptococci) on day 4, and high acidogenicity after 48 h and were susceptible to chlorhexidine at different time intervals. In conclusion, both dentin and saliva can be used as an inoculum in in vitro studies of processes related to biofilm formation.

Caspofungin and Polymyxin B Reduce the Cell Viability and Total Biomass of Mixed Biofilms of Carbapenem-Resistant Pseudomonas aeruginosa and Candida spp.

Pseudomonas aeruginosa and Candida spp. are biofilm-forming pathogens commonly found colonizing medical devices, being mainly associated with pneumonia and bloodstream infections. The coinfection by these pathogens presents higher mortality rates when compared to those caused by a single microbial species. This study aimed to evaluate the antibiofilm activity of echinocandins and polymyxin B (PMB) against polymicrobial biofilms of carbapenem-resistant (CR) Pseudomonas aeruginosa and Candida spp. (C. albicans, C. parapsilosis, C. tropicalis, and C. glabrata). In addition, we tested the antimicrobial effect on their planktonic and monomicrobial biofilm counterparties. Interestingly, beyond inhibition of planktonic [minimum inhibitory concentration (MIC) = 0.5 µg/ml] and biofilm [minimum biofilm inhibitory concentration (MBIC)50 ≤ 2-8 µg/ml] growth of P. aeruginosa, PMB was also effective against planktonic cells of C. tropicalis (MIC = 2 µg/ml), and polymicrobial biofilms of CR P. aeruginosa with C. tropicalis (MBIC50 ≤ 2 µg/ml), C. parapsilosis (MBIC50 = 4-16 µg/ml), C. glabrata (MBIC50 = 8-16 µg/ml), or C. albicans (MBIC50 = 8-64 µg/ml). On the other hand, while micafungin (MFG) showed highest inhibitory activity against planktonic (MIC ≤ 0.008-0.5 µg/ml) and biofilm (MBIC50 ≤ 2-16 µg/ml) growth of Candida spp.; caspofungin (CAS) displays inhibitory activity against planktonic cells (MIC = 0.03-0.25 µg/ml) and monomicrobial biofilms (MBIC50 ≤ 2-64 µg/ml) of Candida spp., and notably on planktonic and monomicrobial biofilms of CR P. aeruginosa (MIC or MBIC50 ≥ 64 µg/ml). Particularly, for mixed biofilms, while CAS reduced significantly viable cell counts of CR P. aeruginosa and Candida spp. at ≥32 and ≥ 2 µg/ml, respectively; PMB was effective in reducing viable cells of CR P. aeruginosa at ≥2 µg/ml and Candida spp. at ≥8 µg/ml. Similar reduction of viable cells was observed for CAS (32-64 µg/ml) combined with PMB (2 µg/ml). These findings highlight the potential of PMB and CAS for the treatment of polymicrobial infections caused by Candida spp. and critical priority CR P. aeruginosa.

Extrato de Rosmarinus Officinalis l. (Alecrim) reduz a viabilidade de biofilmes de interesse para saúde bucal / Rosmarinus officinalis l. (Rosemary) extract decreases the biofilms viability of oral health interest

Objetivo: No presente estudo foi avaliado o efeito do extrato de alecrim sobre a viabilidade de biofilmes monomicrobianos de Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans e Pseudomonas aeruginosa, bem como, sobre biofilmes polimicrobianos de C. albicans associada com S. aureus, E. faecalis, S. mutans ou P. aeruginosa. Material e métodos: Em placa de microtitulação foram formados os biofilmes mono e polimicrobianos por 48 h. Em seguida, foram expostos por 5 min ao extrato de alecrim (200 mg/mL). Solução salina (NaCl 0,9%) foi utilizada como controle. Após, foram realizadas lavagens com salina para remoção de células não aderidas. Para verificação da viabilidade dos biofilmes, após o tratamento, foi aplicado o teste colorimétrico MTT. A absorbância dos poços foi lida em espectrofotômetro de microplacas (570 nm) e os dados foram convertidos em percentual de redução e analisados estatisticamente por ANOVA e Tukey Test (P ≤ 0,05). Resultados: Após aplicação do extrato de alecrim, com exceção do biofilme de E. faecalis, foram observadas reduções significativas da viabilidade dos biofilmes monomicrobianos e polimicrobianos. Conclusão: Biofilmes monomicrobianos de C. albicans, S. aureus, S. mutans e P. aeruginosa, foram afetados pelo extrato de alecrim, bem como, os biofilmes polimicrobianos de C. albicans associada com S. aureus, E. faecalis, S. mutans ou P. aeruginosa em biofilmes polimicrobianos, apresentando significativas reduções de viabilidade.(AU)

Objective: This study evaluated the effect of rosemary extract on Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans and Pseudomonas aeruginosa monomicrobial biofilms viability, as well as on C. albicans associated with S. aureus, E. faecalis, S. mutans or P. aeruginosa in polymicrobial biofilms. Material and Methods: In microtiter plate, mono- and polymicrobial biofilms for 48 h were formed. Then, they were exposed for 5 min to rosemary extract (200 mg/mL). Saline (0.9% NaCl) was used as control. After, washes were done with saline to remove the non-adhered cells. Biofilm viability was checked by MTT colorimetric assay, after treatment. Absorbance of the wells was read in microplate spectrophotometer (570 nm) and data were converted to reduction percentage and statistically analyzed by ANOVA and Tukey test (P ≤ 0.05). Results: After application of rosemary extract, with exception of the E. faecalis biofilm, significant reductions in mono- and polymicrobial biofilms viability were observed. Conclusion: C. albicans, S. aureus, S. mutans and P. aeruginosa monomicrobial biofilms were affected by rosemary extract, as well as C. albicans associated with S. aureus, E. faecalis, S. mutans or P. aeruginosa in polymicrobial biofilms, presenting significant viability reductions. (AU)

New frontiers for anti-biofilm drug development.

Pathogenic microbial biofilm, a consortium of microbial cells protected by a self-produced polymer matrix, is considered a worldwide challenge due to the inherent antibiotic resistance conferred by its lifestyle. Living, as it does, in a community of microbial organisms in a clinical situation, makes it responsible for severe and dangerous cases of infection. Combating this organisation of cells usually requires high antibiotic doses for a prolonged time, and these approaches often fail, contributing to infection persistence. In addition to therapeutic limitations, biofilms can be a source of infections when they grow in medical devices. The challenge imposed by biofilms has mobilised researchers in the entire world to prospect or develop alternatives to control biofilms. In this context, this review summarises the new frontiers that could be used in clinical circumstances in order to prevent or eliminate pathogenic biofilms.