Advancing Anti-Biofilm Strategies: Innovations to Combat Biofilm-Related Challenges and Enhance Efficacy.

Biofilms are complex communities of microorganisms that can cause significant challenges in various settings, including industrial processes, environmental systems, and human health. The protective nature of biofilms makes them resistant to traditional anti-biofilm strategies, such as chemical agents, mechanical interventions, and surface modifications. To address the limitations of conventional anti-biofilm methods, researchers have explored emerging strategies that encompass the use of natural compounds, nanotechnology-based methods, quorum-sensing inhibition, enzymatic degradation, and antimicrobial photodynamic/sonodynamic therapy. There is an increasing focus on combining multiple anti-biofilm strategies to combat resistance and enhance effectiveness. Researchers are continuously investigating the mechanisms of biofilm formation and developing innovative approaches to overcome the limitations of conventional anti-biofilm methods. These efforts aim to improve the management of biofilms and prevent infections while preserving the environment. This study provides a comprehensive overview of the latest advancements in anti-biofilm strategies. Given the dynamic nature of this field, exploring new approaches is essential to stimulate further research and development initiatives. The effective management of biofilms is crucial for maintaining the health of industrial processes, environmental systems, and human populations.

Antimicrobial activity of D-amino acid in combination with photo-sonoactivated hypericin nanoparticles against Acinetobacter baumannii.

BACKGROUND: The emergence of multidrug-resistant Acinetobacter baumannii strains is increasing worldwide. To overcome these life-threatening infections, the development of new treatment approaches is critical. For this purpose, this study was conducted to determine the antimicrobial photo-sonodynamic therapy (aPSDT) using hypericin nanoparticles (HypNP) in combination with D-Tryptophan (D-Trp) against A. baumannii. MATERIALS AND METHODS: HypNP was synthesized and characterized, followed by the determination of the fractional inhibitory concentration (FIC) index of HypNP and D-Trp by checkerboard assay. Next, the antimicrobial and anti-biofilm potential of HypNP@D-Trp-mediated aPSDT against A. baumannii was evaluated. Finally, the anti-virulence activity of aPSDT using HypNP@D-Trp was accessed following the characterization of HypNP@D-Trp interaction with AbaI using in silico virtual screening and molecular docking. RESULTS: A synergistic activity in the combination of HypNP and D-Trp against A. baumannii was observed with a FIC index value of 0.5. There was a 5.10 log10 CFU/mL reduction in the cell viability of A. baumannii when the bacterial cells were treated with 1/2 × MIC of HypNP@D-Trp and subsequently exposed to ultrasound waves and blue light (P < 0.05). Moreover, a significant biofilm degradation effect on biofilm-associated cells of A. baumannii was observed after treatment with aPSDT using 2 × MIC of HypNP@D-Trp in comparison with the control groups (P < 0.05). According to the molecular docking analysis of the protein-ligand complex, Hyp with a high affinity for AbaI showed a binding affinity of - 9.41 kcal/mol. Also, the expression level of abaI gene was significantly downregulated by 10.32-fold in A. baumannii treated with aPSDT as comprised with the control group (P < 0.05). CONCLUSIONS: It can be concluded that HypNP@D-Trp-mediated aPSDT can be considered a promising strategy to overcome the infections caused by A. baumannii by reducing the growth of bacterial biofilm and decreasing the expression of abaI as a gene involved in A. baumannii biofilm formation.

Contribution of antimicrobial photo-sonodynamic therapy in wound healing: an in vivo effect of curcumin-nisin-based poly (L-lactic acid) nanoparticle on Acinetobacter baumannii biofilms.

BACKGROUND: The biofilm-forming ability of Acinetobacter baumannii in the burn wound is clinically problematic due to the development of antibiotic-resistant characteristics, leading to new approaches for treatment being needed. In this study, antimicrobial photo-sonodynamic therapy (aPSDT) was used to assess the anti-biofilm efficacy and wound healing activity in mice with established A. baumannii infections. METHODS: Following synthesis and confirmation of Curcumin-Nisin-based poly (L-lactic acid) nanoparticle (CurNisNp), its cytotoxic and release times were evaluated. After determination of the sub-significant reduction (SSR) doses of CurNisNp, irradiation time of light, and ultrasound intensity against A. baumannii, anti-biofilm activity and the intracellular reactive oxygen species (ROS) generation were evaluated. The antibacterial and anti-virulence effects, as well as, histopathological examination of the burn wound sites of treated mice by CurNisNp-mediated aPSDTSSR were assessed and compared with silver sulfadiazine (SSD) as the standard treatment group. RESULTS: The results showed that non-cytotoxic CurNisNp has a homogeneous surface and a sphere-shaped vesicle with continuous release until the 14th day. The dose-dependent reduction in cell viability of A. baumannii was achieved by increasing the concentrations of CurNisNp, irradiation time of light, and ultrasound intensity. There was a time-dependent reduction in biofilm growth, changes in gene expression, and promotion in wound healing by the acceleration of skin re-epithelialization in mice. Not only there was no significant difference between aPSDTSSR and SSD groups in antibacterial and anti-virulence activities, but also wound healing and re-epithelialization occurred more efficiently in aPSDTSSR than in the SSD group. CONCLUSIONS: In conclusion, CurNisNp-mediated aPSDT might be a promising complementary approach to treat burn wound infections.

Application of antimicrobial sonodynamic therapy as a potential treatment modality in dentistry: A literature review.

The accumulation of dental plaque is a precursor to various dental infections, including lesions, inflammation around dental implants, and inflammation under dentures. Traditional cleaning methods involving physical removal and chemical agents often fall short of eliminating bacteria and their protective biofilms. These methods can also inadvertently lead to bacteria that resist drugs and upset the mouth's microbial harmony. To counter these issues, a new approach is needed that can target and clear away dental plaque, minimize biofilms and bacteria, and thus support sustained dental health. Enter antimicrobial sonodynamic therapy (aSDT), a supplementary treatment that uses gentle ultrasound waves to trigger a sonosensitizer compound, destroying bacterial cells. This process works by generating heat, mechanical pressure, initiating chemical reactions, and producing reactive oxygen species (ROS), offering a fresh tactic for managing dental plaque and biofilms. The study reviews how aSDT could serve as an innovative dental treatment option to enhance oral health.

New strategies to enhance antimicrobial photo-sonodynamic therapy based on nanosensitizers against bacterial infections.

The rapid evolution and spread of multidrug resistance among bacterial pathogens has significantly outpaced the development of new antibiotics, underscoring the urgent need for alternative therapies. Antimicrobial photodynamic therapy and antimicrobial sonodynamic therapy have emerged as promising treatments. Antimicrobial photodynamic therapy relies on the interaction between light and a photosensitizer to produce reactive oxygen species, which are highly cytotoxic to microorganisms, leading to their destruction without fostering resistance. Antimicrobial sonodynamic therapy, a novel variation, substitutes ultrasound for light to activate the sonosensitizers, expanding the therapeutic reach. To increase the efficiency of antimicrobial photodynamic therapy and antimicrobial sonodynamic therapy, the combination of these two methods, known as antimicrobial photo-sonodynamic therapy, is currently being explored and considered a promising approach. Recent advances, particularly in the application of nanomaterials, have further enhanced the efficacy of these therapies. Nanosensitizers, due to their improved reactive oxygen species generation and targeted delivery, offer significant advantages in overcoming the limitations of conventional sensitizers. These breakthroughs provide new avenues for treating bacterial infections, especially multidrug-resistant strains and biofilm-associated infections. Continued research, including comprehensive clinical studies, is crucial to optimizing nanomaterial-based antimicrobial photo-sonodynamic therapy for clinical use, ensuring their effectiveness in real-world applications.

Evaluation of anti-biofilm effect of antimicrobial sonodynamic therapy-based periodontal ligament stem cell-derived exosome-loaded kojic acid on Enterococcus faecalis biofilm.

Introduction. Antimicrobial sonodynamic therapy (aSDT) is an approach that uses ultrasound waves (UWs) and a sonosensitizer to generate reactive oxygen species (ROS) to damage microbial cells in biofilms. Using nano-carriers, such as exosomes (Exos), to deliver the sonosensitizer can potentially enhance the effectiveness of aSDT.Hypothesis/Gap Statement. aSDT can downregulate the expression of gelE and sprE genes, increasing the production of endogenous ROS and degradation of pre-formed Enterococcus faecalis biofilms.Aim. This study investigated the anti-biofilm effect of aSDT-based periodontal ligament stem cell-derived exosome-loaded kojic acid (KA@PDL-Exo) on pre-formed E. faecalis biofilms in root canals.Methodology. Following the isolation and characterization of PDL-Exo, KA@PDL-Exo was prepared and confirmed. The minimal biofilm inhibitory concentration (MBIC) of KA, PDL-Exo, KA@PDL-Exo and sodium hypochlorite (NaOCl) was determined, and their anti-biofilm effects were assessed with and without UWs. The binding affinity of KA with GelE and SprE proteins was evaluated using in silico molecular docking. Additionally, the study measured the generation of endogenous ROS and evaluated changes in the gene expression levels of gelE and sprE.Results. The results revealed a dose-dependent decrease in the viability of E. faecalis cells within biofilms. KA@PDL-Exo was the most effective, with an MBIC of 62.5 µg ml-1, while NaOCl, KA and PDL-Exo had MBIC values of 125, 250 and 500 µg ml-1, respectively. The use of KA@PDL-Exo-mediated aSDT resulted in a significant reduction of the E. faecalis biofilm (3.22±0.36 log10 c.f.u. ml-1; P<0.05). The molecular docking analysis revealed docking scores of -5.3 and -5.2 kcal mol-1 for GelE-KA an SprE-KA, respectively. The findings observed the most significant reduction in gene expression of gelE and sprE in the KA@PDL-Exo group, with a decrease of 7.9- and 9.3-fold, respectively, compared to the control group (P<0.05).Conclusion. The KA@PDL-Exo-mediated aSDT was able to significantly reduce the E. faecalis load in pre-formed biofilms, decrease the expression of gelE and srpE mRNA, and increase the generation of endogenous ROS. These findings imply that KA@PDL-Exo-mediated aSDT could be a promising anti-biofilm strategy that requires additional in vitro and in vivo investigations.

Enhancement of hypericin nanoparticle-mediated sonoinduced disruption of biofilm and persister cells of Streptococcus mutans by dermcidin-derived peptide DCD-1L.

BACKGROUND: Streptococcus mutans is considered a major significant contributor to dental caries and its effective removal is difficult due to the formation of biofilm. Therefore, the development of adjuvant therapeutic strategies with anti-biofilm properties is a promising approach. In the present study, we examined the effect of dermcidin-derived peptide DCD-1 L on the antibacterial activity of hypericin nanoparticle (HypNP)-mediated antimicrobial sonodynamic therapy (aSDT) against persister cells growing- and biofilm cultures of S. mutans. MATERIALS AND METHODS: Following synthesis and confirmation of HypNP, the fractional inhibitory concentration (FIC) index of HypNP and DCD-1 L was determined by checkerboard assay. Cellular uptake of HypNP-DCD-1 L and generation of endogenous reactive oxygen species (ROS) were assessed and followed by the determination of antimicrobial sonoactivity of HypNP-DCD-1 L against persister cells growing- and biofilm cultures of S. mutans. The water-insoluble extracellular polysaccharide (EPS) and expression of the gtfD, comDE, and smuT genes were then evaluated in persister cells growing- and biofilm cultures of S. mutans. RESULTS: There was a synergistic activity in the combination of HypNP and DCD-1 L against S. mutans with an FIC index value of 0.37. The HypNP-DCD-1L-mediated aSDT also displayed the highest cellular uptake and endogenous ROS generation by bacterial cells. When biofilm and persister cells of S. mutans were treated with HypNP-DCD-1 L and subsequently exposed to ultrasound waves, 5.1 log and 3.8 log reductions, respectively, in bacterial numbers were observed (P<0.05). According to the data, EPS in both persister cells growing- and biofilm cultures of S. mutans were significantly decreased after exposure to the HypNP-DCD-1L-mediated aSDT (P<0.05). In addition, the quantitative real-time PCR data illustrated the high level of similarities in very low-expression profiles of the gtfD before and after all treated groups for persister cells. While, following HypNP-DCD-1L-mediated aSDT treatment, the expression levels of gtfD, comDE, and smuT were significantly lower in treated persister cells growing- and biofilm cultures of S. mutans in comparison with control groups (P<0.05). CONCLUSIONS: Combined, the results of this study indicate that ultrasound waves-activated HypNP-DCD-1 L can sonoinactivate S. mutans biofilms and persister cells, as well as reduce effectively pathogenicity potency of S. mutans. Hence, HypNP-DCD-1L-mediated aSDT may be proposed as a promising adjunctive therapeutic approach for dental caries.

Anti-biofilm and bystander effects of antimicrobial photo-sonodynamic therapy against polymicrobial periopathogenic biofilms formed on coated orthodontic mini-screws with zinc oxide nanoparticles.

BACKGROUND: The present study evaluated the anti-biofilm and bystander effects of antimicrobial photo-sonodynamic therapy (aPSDT) on the polymicrobial periopathogenic biofilms formed on mini-screws coated with zinc oxide nanoparticles (ZnONPs). MATERIALS AND METHODS: Thirty orthodontic identical mini-screws were divided into 6 groups (n = 5) as follows: 1. negative control: uncoated mini-screw + phosphate-buffered saline (PBS), 2. positive control: uncoated mini-screw + 0.2% CHX, 3. coating control: coated mini-screw + PBS, 4. antimicrobial photodynamic therapy (aPDT): coated mini-screw+light emitting diode (LED), 5. Antimicrobial sonodynamic therapy (aSDT): coated mini-screw+ultrasound waves, and 6. aPSDT: coated mini-screw+LED+ultrasound waves. Electrostatic spray-assisted vapor deposition was employed to coat ZnONPs on titanium mini-screws. The biofilm inhibition test was used to assess the anti-biofilm efficacy against polymicrobial periopathogenic biofilms including Porphyromonas gingivitis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans, and the results were shown as the percent reduction of Log10 colony-forming unit (CFU)/mL. Following each treatment, the gene expression levels of TNF-α, IL-1ß, and IL-6 were evaluated on human gingival fibroblast (HGF) cells via quantitative real-time polymerase chain reaction (qRT-PCR) to reveal the bystander effects of aPSDT on HGF cells. RESULTS: A significant reduction in log10 CFU/mL of periopathogens was observed in groups treated with aPDT, aSDT, aPSDT, and 0.2% CHX up to 6.81, 6.63, 5.02, and 4.83 log, respectively, when compared with control groups (P<0.05). 0.2% CHX and aPSDT groups demonstrated significantly higher capacity in eliminating the periopathogen biofilm compared with other groups (P<0.05). The qRT-PCR showed that the expression level of inflammatory cytokines was significantly down regulated in aPDT, aSDT, and aPSDT groups (P<0.05). CONCLUSION: It was found that the ZnONPs-mediated aPSDT could significantly reduce periopathogen biofilm as well as the expression level of inflammatory cytokines.

Anti-multispecies microbial biofilms and anti-inflammatory effects of antimicrobial photo-sonodynamic therapy based on acrylic resin containing nano-resveratrol.

BACKGROUND: Polymethylmethacrylate (PMMA)-based removable orthodontic appliances are susceptible to microbial colonization due to the surface porosity, and accumulating the biofilms causes denture stomatitis. the present study evaluated the anti-biofilm and antiinflammatory effects of antimicrobial photo-sonodynamic therapy (aPSDT) against multispecies microbial biofilms (Candida albicans, Staphylococcus aureus, Streptococcus sobrinus, and Actinomyces naeslundii) formed on acrylic resin modified with nanoresveratrol (NR). MATERIALS AND METHODS: Following the determination of the minimum biofilm inhibitory concentration (MBIC) of NR, in vitro anti-biofilm activity of NR was evaluated. The antibiofilm efficacy against multispecies microbial biofilm including C. albicans, S. aureus, S. sobrinus, and A. naeslundii, were assessed by biofilm inhibition test and the results were measured. To reveal the anti-inflammatory effects of aPSDT on human gingival fibroblast (HGF) cells, the gene expression levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were evaluated via quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: According to the results, the MBIC dose of NR against multispecies microbial biofilm was considered 512 µg/mL. The highest biofilm reduction activity was observed in MBIC treated with aPSDT and 2 × MBIC exposed to light emitting diode (LED) and ultrasound waves (UW). The expression level of TNF-α and IL-6 genes were significantly increased when HGF cells were exposed to multispecies microbial biofilms (P<0.05), while after treatment with aPSDT, the expression levels of genes were significantly downregulated in all groups (P<0.05). CONCLUSION: Overall, NR-mediated aPSDT reduced the growth of the multispecies microbial biofilm and downregulated the expression of TNF-α and IL-6 genes. Therefore, modified PMMA with NR can be serving as a promising new orthodontic acrylic resin against multispecies microbial biofilms and the effect of this new material is amplified when exposed to LED and UW.

Ex vivo efficacy of sonodynamic antimicrobial chemotherapy for inhibition of Enterococcus faecalis and Candida albicans biofilm.

BACKGROUND: This study aimed to assess the ex vivo efficacy of sonodynamic antimicrobial chemotherapy (SACT) also known as antimicrobial sonodynamic therapy for inhibition of Enterococcus faecalis and Candida albicans biofilm. MATERIALS AND METHODS: This study was conducted on 80 extracted single-canal maxillary anterior teeth. After instrumentation, the root canals were inoculated with E. faecalis and C. albicans suspensions, and the teeth were assigned to 5 groups of control (no antimicrobial therapy), nano-curcumin, ultrasound waves, 5.25% sodium hypochlorite (NaOCl), and SACT (nano-curcumin plus ultrasound waves). The mean biofilm thickness and number of colonies were then counted. RESULTS: The E. faecalis colony count in nano-curcumin, ultrasound waves, and SACT groups was significantly lower than that in the control group (P<0.05). The C. albicans colony count in SACT group was significantly lower than that in the control and ultrasound waves groups (P<0.05). The mean biofilm thickness in NaOCl and SACT groups was significantly thinner than other groups (P<0.05). The mean biofilm thickness in SACT group was significantly thinner than that in ultrasound waves group (P<0.001). CONCLUSION: In summary, SACT using nano-curcumin had an almost comparable efficacy to NaOCl, but was more effective than ultrasound waves and nano-curcumin for reduction of C. albicans and E. faecalis biofilm.

An ex vivo evaluation of physico-mechanical and anti-biofilm properties of resin-modified glass ionomer containing ultrasound waves-activated nanoparticles against Streptococcus mutans biofilm around orthodontic bands.

BACKGROUND: The present study evaluated the physico-mechanical and antimicrobial properties of ultrasound waves-activated modified-resin glass ionomer containing nanosonosensitizers such as nano-curcumin (n-Cur), nano-emodin (n-Emo), and nano-quercetin (n-Qct) against Streptococcus mutans biofilm on the surface of modified-resin glass ionomer bonded orthodontic bands. MATERIALS AND METHODS: A total of 50 human molar teeth were used in this study. The shear bond strength (SBS), adhesive remnant index (ARI), setting time, and fluoride release of modified orthodontics cement containing different concentrations of n-Cur, n-Emo, and n-Qct (0, 2, 5, and 10%) were measured. The antimicrobial effectiveness was assessed against S. mutans by the biofilm inhibition test, and the Log10 colony-forming unit (CFU)/mL was evaluated. RESULTS: SBS and setting time of modified glass ionomer decreased compared with the control group. 5% n-Emo, 2% n-Qct, and 5% n-Cur were the highest concentrations that had an insignificant difference in comparison with Transbond XT (P = 0.647, 0.819, and 0.292, respectively). The groups were not significantly different in terms of ARI score (P > 0.05). The highest and lowest setting time belonged to the control and 5% n-Emo groups, respectively; this difference in setting time was significant (P < 0.05). Ultrasound waves and 0.2% CHX significantly reduced S. mutans biofilms compared with the control group (P < 0.001), and minimum S. mutans colony count was shown in 0.2% CHX and 5% n-Emo groups. The addition of nanosonosensitizers to the glass ionomer did not compromise the fluoride release of the glass ionomer. CONCLUSION: It could be concluded that resin-modified glass ionomer containing ultrasound waves-activated 5% n-Emo reduces S. mutans biofilm around orthodontic bands with no adverse effect on SBS, ARI, and its application in the clinic.

Synergistic biocidal effects of metal oxide nanoparticles-assisted ultrasound irradiation: Antimicrobial sonodynamic therapy against Streptococcus mutans biofilms.

BACKGROUND: Antimicrobial sonodynamic therapy (aSDT) is an adjunctive modality, which uses ultrasound irradiation to kill microbial cells by the activation of a sonosensitizer. The aim of this study was to evaluated the synergistic biocidal effects of zinc oxide nanoparticles (ZnO NPs) and titanium dioxide nanoparticles (TiO2 NPs) as the metal oxide nanoparticles (MONPs)-assisted ultrasound irradiation against Streptococcus mutans biofilms. MATERIALS AND METHODS: Following preparation and characterization of MONPs, cellular uptake and generation of intracellular reactive oxygen species (ROS) were assessed. After determination of the sub-significant reduction (SSR) doses of ZnO NPs, TiO2 NPs, ZnO/TiO2 NPs, and ultrasound intensity against S. mutans, anti-biofilm effects of aSDT were assessed using colorimetric assay, plate counting, and field emission scanning electron microscope (FESEM) analysis. Also, the metabolic activity of S. mutans and the expression levels of glucosyltransferase B (gtfB) as a main virulence factor of S. mutans were evaluated by XTT assay and quantitative real-time polymerase chain reaction following ZnO/TiO2 NPsSSR- mediated aSDT. RESULTS: The finding of this study showed that an incubation time of 5 min was sufficient to achieve maximal uptake of MONPs. The ROS production following aSDT using ZnO NPs, TiO2 NPs, and ZnO/TiO2 NPs were ~ 4.1-, 5.6-, and 11.7-fold increase, respectively. The dose-dependent reduction in cell viability of S. mutans was revealed by increasing the concentrations of ZnO NPs, TiO2 NPs, ZnO/TiO2, as well as ultrasound intensities. According to the data, 1.5 µg/mL, 3.1 µg/mL, 25 µg/mL, and 0.75 W/cm2 were considered as the SSR doses of ZnO/TiO2 NPs, ZnO NPs, TiO2 NPs, and ultrasound intensity, respectively (P>0.05). ZnO/TiO2 NPsSSR-mediated aSDT showed a significantly higher biofilm inhibitory activity than the other treatment groups (P<0.05). Based on the FE-SEM analysis, aSDT based on the ZnO/TiO2 NPsSSR had a strong anti-biofilm effect against preformed biofilms of S. mutans on the enamel slabs. Also, the metabolic activity of S. mutans and the expression levels of gtfB were significantly decreased to 85.5% and 12.3-fold, respectively following ZnO/TiO2 NPsSSR-mediated aSDT (P<0.05). No considerable difference was observed in anti-biofilm activity between ZnO/TiO2 NPsSSR- mediated aSDT and 0.2% CHX (P>0.05). CONCLUSION: The results revealed anti-metabolic and anti-biofilm potential activities of ZnO/TiO2 NPs-mediated aSDT against S. mutans with the highest cellular uptake and ROS generation.