Biofilm Matrix Research Articles

The DAIR-procedure in fracture-related infection-When and how.

This narrative review discusses treatment strategies and key considerations guiding decisions in the surgical management of fracture-related infections (FRI). Treatment options primarily revolve around two approaches: debridement antibiotics and implant retention (DAIR), or implant exchange, either in a one or multiple-stage strategy. Several considerations, including time since infection onset, implant type, stability, causative pathogens, host physiology, and soft tissue conditions, inform the choice of surgical intervention for FRI. Current literature supports the preference for a DAIR approach in cases with a short duration of ongoing symptoms, a stable implant with satisfactory fracture reduction, and a viable soft tissue envelope. Conversely, one- or multiple-stage implant exchange is deemed beneficial in instances of compromised local and systemic host physiology, mature biofilm, difficult-to-treat pathogens, intramedullary implants, and cases involving reinfections or failed DAIR procedures. Notably, these recommendations draw parallels from periprosthetic joint infection treatment strategies, constrained by the limited availability of randomized controlled trials comparing these options specifically in acute FRI. In conclusion, future perspectives call for extensive investigations into biofilm maturation and the impact of time on treatment outcomes. Additionally, there is a need for a standardized classification system for FRI to enhance the comparability of treatment outcomes in FRI management.

Laser NIR Irradiation Enhances Antimicrobial Photodynamic Inactivation of Biofilms of Staphylococcus aureus.

Photodynamic inactivation (PDI) is a powerful technique for eradicating microorganisms, and our group previously demonstrated its effectiveness against planktonic cultures of Staphylococcus aureus bacteria using 5,10,15,20-tetrakis[4-(3-N,N-dimethylaminopropoxy)phenyl]porphyrin (TAPP) and visible light irradiation. However, biofilms exhibit a lower sensitivity to PDI, mainly due to limited penetration of the photosensitizer (PS). In the context of emerging antibacterial strategies, near-infrared treatments (NIRTs) have shown promise, especially for combating resistant strains. NIRT can act either through photon absorption by water, causing a thermal effect on bacteria, or by specific chromophores without a significant temperature increase. Our objective was to enhance biofilm sensitivity to TAPP-PDI by pretreatment with NIRT. This combined approach aims to disrupt biofilms and increase the efficacy of TAPP-PDI against bacterial biofilms. In vitro biofilm models of S. aureus RN6390 were utilized. NIRTs involved a 980 nm laser (continuous mode, 7.5 W/cm2, 30 s, totaling 225 J/cm2) post-TAPP exposure to enhance photosensitizer accumulation. Subsequent visible light irradiation at 180 J/cm2 was employed to perform PDI. Colony-forming unit counts evaluated the synergistic effect on bacterial viability. Scanning electron microscopy visualized the architectural changes in the biofilm structure. TAPP was extracted from bacteria to estimate the impact of NIRT on biofilm penetration. Using in vitro biofilm models, NIRT application following biofilm exposure to TAPP increased PS accumulation per bacteria. Under these conditions, NIRT induced a transient increase in the temperature of PBS to 46.0 ± 2.6°C (ΔT = 21.5°C). Following exposure to visible light, a synergistic effect emerged, yielding a substantial 4.4 ± 0.1-log CFU reduction. In contrast, the PDI and NIRT treatments individually caused a decrease in viability of 0.9 ± 0.1 and 0.8 ± 0.2-log respectively. Interestingly, preheating TAPP-PBS to 46°C had no significant impact on TAPP-PDI efficacy, suggesting the involvement of thermal and nonthermal effects of NIR action. In addition to the enhanced TAPP penetration, NIRT dispersed the biofilms and induced clefts in the biofilm matrix. Our findings suggest that NIR irradiation serves as a complementary treatment to PDI. This combined strategy reduces bacterial numbers at lower PS concentrations than standalone PDI treatment, highlighting its potential as an effective and resource-efficient antibacterial approach.

Antifungal properties of Abnormal Cannabinoid derivatives: Disruption of Biofilm Formation and Gene Expression in Candida Species

Abnormal cannabinoids (including comp 3) are a class of synthetic lipid compounds with non-psychoactive properties and regioisomer configurations, but distinct from traditional cannabinoids since they do not interact with the established CB1 and CB2 receptors. Previous research showed the cardioprotective and anti-inflammatory potentials of comp 3 and more recently its antimicrobial effect on methicillin-resistant Staphylococcus aureus (MRSA). Given the escalating challenges posed by Candida infections and the rise of antifungal drug resistance, the exploration of novel therapeutic avenues is crucial. This study aimed to assess the anti-Candida properties of newly synthesized AbnCBD derivatives. AbnCBD derivatives were synthesized by acid catalysis-induced coupling and further derivatized. We evaluated the potential of the AbnCBD derivatives to inhibit the growth stages of various Candida species. By in vitro colorimetric assays and in vivo mice experiments, we have shown that AbnCBD derivatives induce differential inhibition of Candida growth. The AbnCBD derivatives, especially comp 3, comp 10, and comp 9 significantly reduced the growth of C. albicans, including FLC-resistant strains, and of C. tropicalis and C. parapsilosis but not of C auris compared to their controls (FLC and 0.5% DMSO). Comp 3 also disrupted C. albicans biofilm formation and eradicated mature biofilms. Notably, other derivatives of AbnCBD disrupted the biofilm formation and maturation of C. albicans but did not affect yeast growth. In a murine model of VVC, comp 3 demonstrated significant fungal clearance and reduced C. albicans burden compared to vehicle and FLC controls. These findings highlight the potential of AbnCBDs as promising antifungal agents against Candida infections.

Equine bone marrow-derived mesenchymal stromal cells reduce established S. aureus and E. coli biofilm matrix in vitro.

Biofilms reduce antibiotic efficacy and lead to complications and mortality in human and equine patients with orthopedic infections. Equine bone marrow-derived mesenchymal stromal cells (MSC) kill planktonic bacteria and prevent biofilm formation, but their ability to disrupt established orthopedic biofilms is unknown. Our objective was to evaluate the ability of MSC to reduce established S. aureus or E. coli biofilms in vitro. We hypothesized that MSC would reduce biofilm matrix and colony-forming units (CFU) compared to no treatment and that MSC combined with the antibiotic, amikacin sulfate, would reduce these components more than MSC or amikacin alone. MSC were isolated from 5 adult Thoroughbred horses in antibiotic-free medium. 24-hour S. aureus or E. coli biofilms were co-cultured in triplicate for 24 or 48 hours in a transwell plate system: untreated (negative) control, 30 μg/mL amikacin, 1 x 106 passage 3 MSC, and MSC with 30 μg/mL amikacin. Treated biofilms were photographed and biofilm area quantified digitally. Biomass was quantified via crystal violet staining, and CFU quantified following enzymatic digestion. Data were analyzed using mixed model ANOVA with Tukey post-hoc comparisons (p < 0.05). MSC significantly reduced S. aureus biofilms at both timepoints and E. coli biofilm area at 48 hours compared to untreated controls. MSC with amikacin significantly reduced S. aureus biofilms versus amikacin and E. coli biofilms versus MSC at 48 hours. MSC significantly reduced S. aureus biomass at both timepoints and reduced S. aureus CFU at 48 hours versus untreated controls. MSC with amikacin significantly reduced S. aureus biomass versus amikacin at 24 hours and S. aureus and E. coli CFU versus MSC at both timepoints. MSC primarily disrupted the biofilm matrix but performed differently on S. aureus versus E. coli. Evaluation of biofilm-MSC interactions, MSC dose, and treatment time are warranted prior to testing in vivo.

Antioxidant: Antimycobacterial and Antibiofilm Activities of Acetone Extract and Subfraction Artemisia afra Jacq. ex Willd. Against Mycobacterium smegmatis

Tuberculosis is a worldwide prevalent and recurring disease that contributes significantly to high mortality rates. This study aimed to investigate the antioxidant, anti-mycobacterial, and antibiofilm activities of Artemisia afra acetone crude extract. Methodology: The crude acetone extract was fractionated using column chromatography and characterized by liquid chromatography–mass spectroscopy (LC-MS). A 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was used to assess the antioxidant activity. The antimycobacterial activity against Mycobacterium smegmatis was screened using bioautography, broth microdilution, and growth curve assays. Molecular docking was used to predict the possible mechanisms of action of the LC-MS-identified ligands. Crystal violet was used to screen for anti-cell adherence and biofilm inhibition activities. Results: The crude extract scavenged 77% of the free radical at 16 μg/mL. The subfraction had a lower minimum inhibitory concentration (MIC) (0.078 mg/mL) compared to the crude extract (0.313–0.833 mg/mL). The subfraction had a concentration-dependent inhibition effect (&gt;50%) on mycobacterial cell adherence and early biofilms. However, the mature biofilms were resistant. Two propanoate compounds, [(2S)-3-[6-acetyl-4,6-dihydroxy-3-[(1R)-1-hydroxyethyl]tetrahydropyran-2-yl]-2-hydroxy-propyl] (2R)-2-amino-3-(1H-imidazol-5-yl)propanoate and 3-(6-aminopurin-9-yl)propyl 3-(2,4-dioxo-1,3-diazaspiro[4.5]decan-3-yl) propanoate, had binding energies of −5.4 kcal/mol and −6.3 kcal/mol, respectively, against the RNA polymerase binding protein. Conclusions: The results show that A. afra acetone crude extract has antioxidant and antimycobacterial activities that can be improved by fractionation.

Dietary carbohydrates modulate Streptococcus mutans adherence and bacterial proteome.

Streptococcus mutans adherence to the tooth surface and subsequent biofilm development is modulated by the carbohydrate source, but the corresponding effect on bacterial proteome has not been previously studied. This study aimed to assess the effect of different carbohydrates on S. mutans viability and bacterial proteome at two-time points, early attachment (8 h) and biofilm maturation (24 h). Hydroxyapatite (HAp) discs coated with parotid saliva proteins were inoculated with S. mutans UA159 in Tryptone Soy Broth without dextrose supplemented with one of the following carbohydrates (n=12/treatment/time point): 1% Sucrose (S); 0.525% Glucose + 0.525% Fructose (G+F); 10% Xylitol (X); 10% Xylitol + 1% Sucrose (X+S); or culture medium without supplementation as negative control (C). Once inoculated, HAp discs were incubated for 8 h or 24 h at 37 °C and 10% CO2. After each incubation period, adhered bacteria were quantified using the plate-counting method for 6 HAp discs/group, and the remaining 6 HAp discs/group were used to extract bacterial cell wall proteins. Extracted proteins were analyzed using liquid chromatography coupled with mass spectrometry and then classified by their biological process. The study was conducted in three independent assays and the number of bacteria adhered to the HAp discs was determined at each time point and analyzed by two-way ANOVA followed by Bonferroni test (=5%). The results suggest that xylitol significantly repressed bacterial adherence and metabolism at 8 h and 24 h; however, bacterial adherence and metabolism were significantly enhanced when xylitol was combined with sucrose, showing no negative effect on S. mutans at both time points. Bacterial proteome was modulated by the carbohydrate source. The cariogenicity of S. mutans biofilms may be reduced by the alternative sweetener xylitol; however, the combination with fermentable sugars may inhibit such a beneficial effect.

Cariogenic potential of the Streptococcus mutans Cid/Lrg system: an in vivo animal case study.

In our prior study using a dual-species (Streptococcus mutans/Streptococcus gordonii) competitive mouse caries model to investigate the contribution of S. mutans LrgAB to in vivo fitness, S. mutans wild-type and ΔlrgAB mutants consistently outnumbered S. gordonii and had high caries scores, even though the ΔlrgAB mutant is highly sensitive to oxidative stress. To determine whether the highly cariogenic sucrose diet used in the previous study masked the contribution of LrgAB to competitive fitness of S. mutans against S. gordonii, we recapitulated our previous mouse caries experiment with a modification in which 4% sucrose drinking water was replaced with sterile water, hypothesized to decrease the frequency of exposure of mice to sucrose, a determinant in the cariogenicity of S. mutans. Given that both S. mutans ΔlrgAB and ΔcidB mutants are sensitive to oxidative stress and share similar transcriptional profiles, these strains, as well as wild-type UA159, were tested in this modified dual-species mouse caries model. When comparing between groups the colonization within molar dental biofilms of S. mutans strains, ΔlrgAB mutant was at a level similar to the wild type, whereas S. mutans ΔcidB was modestly lower than both wild-type and S. mutans ΔlrgAB. The severity of total sulcal caries in both the ∆cidB and ∆lrgAB mutant infections was significantly lower than that of wild type. These results demonstrate that the Cid/Lrg system aids in S. mutans fitness against S. gordonii and caries potential in vivo, a phenotype likely masked in our previous study by more frequent exposure to sucrose.IMPORTANCEThe development of a mature biofilm on the tooth surface is the central event in the pathogenesis of dental caries, which primarily requires that cariogenic organisms withstand the limited resources or environmental fluctuations experienced in the oral cavity. The sensitive and heterogeneous response of the cid and lrg operons to complex external signals has been hypothesized to trigger differentiation of the Streptococcus mutans biofilm community into distinct functional subpopulations to promote survival and persistence of the S. mutans community when challenged by an unfavorable environment. The study described herein enlightens our understanding of how Cid/Lrg contributes to S. mutans pathogenic potential in vivo (caries development), warranting further research regarding the adaptive role of Cid/Lrg system in human oral biofilms toward the development of anti-caries strategies directed at the Cid/Lrg system.

Biofilm-mediated antibiotic tolerance in Staphylococcus aureus from spinal cord stimulation device-related infections.

Staphylococcus aureus is a predominant cause of infections in individuals with spinal cord stimulation (SCS) devices. Biofilm formation complicates these infections, commonly requiring both surgical and antibiotic treatments. This study explored the biofilm matrix composition and antimicrobial susceptibility of planktonic and biofilm-growing S. aureus isolates from individuals with SCS-related infections. Whole-genome sequencing (WGS) examined genotypes, virulome, resistome, and the pan-genome structure. The study also analyzed biofilm matrix composition, early surface adhesion, hemolytic activity, and antibiotic-susceptibility testing. WGS revealed genetic diversity among isolates. One isolate, though oxacillin susceptible, contained the mecA gene. The median number of virulence factor genes per isolate was 58. All isolates harbored the biofilm-related icaA/D genes. When assessing phenotypic characteristics, all strains demonstrated the ability to form biofilms in vitro. The antimicrobial susceptibility profile indicated that oxacillin, rifampin, and teicoplanin showed the highest efficacy against S. aureus biofilm. Conversely, high biofilm tolerance was observed for vancomycin, trimethoprim/sulfamethoxazole, and levofloxacin. These findings suggest that S. aureus isolates are highly virulent and produce robust biofilms. In cases of suspected biofilm infections caused by S. aureus, vancomycin should not be the primary choice due to its low activity against biofilm. Instead, oxacillin, rifampin, and teicoplanin appear to be more effective options to manage SCS infections.IMPORTANCESCS devices are increasingly used to manage chronic pain, but infections associated with these devices, particularly those caused by Staphylococcus aureus, present significant clinical challenges. These infections are often complicated by biofilm formation, which protects bacteria from immune responses and antibiotic treatments, making them difficult to eradicate. Understanding the genetic diversity, virulence, and biofilm characteristics of S. aureus isolates from SCS infections is critical to improving treatment strategies. Our study highlights the need to reconsider commonly used antibiotics like vancomycin, which shows reduced activity against biofilm-growing cells. Identifying more effective alternatives, such as oxacillin, rifampin, and teicoplanin, provides valuable insight for clinicians when managing biofilm-related S. aureus infections in patients with SCS implants. This research contributes to the growing evidence that biofilm formation is crucial in treating device-related infections, emphasizing the importance of tailoring antimicrobial strategies to the biofilm phenotype.

Cytocompatibility, Antibacterial, and Anti-Biofilm Efficacy of Grape Seed Extract and Quercetin Hydrogels Against a Mature Endodontic Biofilm Ex Vivo Model

Background/Objectives: To assess the cytocompatibility, antibacterial and anti-biofilm efficacy of grape seed extract (GSE) and quercetin hydrogels versus calcium hydroxide (CH) as intracanal medications (ICMs) against an endodontic ex vivo biofilm model. Methods: Single-rooted teeth (n = 50) were prepared and sterilized before being infected with E. faecalis to develop a mature biofilm. They were divided into five equal groups according to the ICM used: G1: medicated with CH paste, G2: medicated with GSE hydrogel, G3: medicated with quercetin hydrogel, G4: positive control group that was infected and not medicated, and G5: negative control group that was neither infected nor medicated. After 1 week, the ICM was removed, and the root canals were cultured to assess the antibacterial efficacy by counting the colony-forming units and the anti-biofilm efficacy by the crystal violet assay. Dead/live bacterial viability was assessed by CFLSM examination, while the cytocompatibility was assessed using the MTT assay. Results: CH had the best antibacterial efficacy, followed by GSE and quercetin hydrogels (p &lt; 0.001). Regarding the anti-biofilm efficacy, GSE was superior, followed by quercetin and CH (p &lt; 0.001). CFLSM examination showed CH and GSE hydrogel to be highly effective in comparison to the positive control (p &lt; 0.0001), with no statistical difference between them (p &gt; 0.05). CH showed significantly higher cell viability percentages using a 500 μg/mL, while quercetin and GSE started to show cell viability &gt; 70% at concentrations of 125 μg/mL and 62.5 μg/mL. Conclusions: CH fulfilled the ideal requirements of ICM as being both antibacterial and non-cytotoxic compared to the other materials tested.

Impact of Biofilms on Surface Properties of Polymethyl Methacrylate (PMMA) Resins.

Poly(methyl methacrylate) (PMMA) resins are widely used in medical and dental applications. Their susceptibility to bacterial biofilm formation poses significant challenges related to material degradation and infection risk. This study investigated the effects of Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) biofilms on PMMA resin surface properties over a 45-day period at 35°C. The study examined various parameters including biofilm adhesion, morphology, surface roughness, hydrophobicity, solid fraction, and zeta potential. PMMA resin specimens were inoculated with bacteria and incubated for 45 days. Biofilm adhesion was visually assessed, while surface characterization was conducted using scanning electron microscopy (SEM), atomic force microscopy (AFM), roughness analysis, contact angle measurements, solid fraction determination, and zeta potential analysis. The P. aeruginosa and S. aureus isolates were selected based on their biofilm-positive characteristics, which were further confirmed using Congo red and biofilm formation assays through crystal violet staining and spectrophotometric analysis. The results demonstrated robust biofilm adhesion on PMMA surfaces. SEM and AFM imaging revealed textured surfaces with elevated structures and depressions within the biofilm matrix. Biofilm-exposed resins exhibited significantly increased roughness (Ra = 164.5 nm, Rq = 169.5 nm) and hydrophobicity (mean angle = 85.5°-90.5°) compared to control samples (Ra = 38-50 nm, angle = 55°). Solid fraction measurements indicated a denser biofilm matrix on exposed resins (0.908) compared to controls (0.65). Additionally, zeta potential values were more negative for biofilm-exposed resins (mean = -84.2 mV) than controls (-45.0 mV). These findings underscore the substantial alterations in PMMA resin surface properties induced by bacterial biofilms, emphasizing the critical need for strategies to prevent biofilm formation and mitigate associated risks in healthcare settings. Future research should focus on developing anti-biofilm coatings or treatments to preserve the integrity and functionality of PMMA materials.

Biofilm formation and drug susceptibility of biofilm Candida spp. clinically isolated from nasopharyngeal cancer patients in Vietnam

Background and Objectives: The biofilm formation has been widely recognized as one of the main mechanisms of antimi- crobial resistance development in microorganisms. However, few studies are focusing on this phenomenon in Candida spp. in clinical settings, especially on immuno-compromised patients. Materials and Methods: In this study, both the rate of biofilm formation in those patients and its drug susceptibility in initial and mature biofilm were assessed using crystal violet assay and dilution method. Results: The results demonstrated that the biofilm formation rate was similar between albicans and non-albicans Candida. However, the biofilm formation capacity was more pronounced in non-albicans Candida, especially, C. glabrata. As expect- ed, there was a significant relationship between biofilm formation and drug resistance. In addition, our study reconfirmed that the age of high concentration of antifungal agents only affected Candida before its biofilm formation regardless of its biofilm formation capacity. In the contrary, once the biofilm was formed even elevated drug concentrations did not show sufficient efficacy, highlighting a need for high dosage at the early stage of treatment for those patients. Conclusion: The results of this study highlighted the importance of using appropriate antifungal agents for Candida treatment before the formation of biofilm.

Biofilm Microenvironment-Sensitive Anti-Virulent and Immunomodulatory Nano-on-Nanodroplets to Combat Refractory Biofilm Infection Through Toxin Neutralization and Phagocytosis.

Biofilm-associated wound infection is principally perceived as the bacterial defense mechanism that hinders antibiotic penetration, causes toxin impairment, and suppresses the immunological responses of the host immune system. Several antibiofilm agents have been developed, but the least of these agents can simultaneously cornerstone on the biofilm-associated immunosuppression and bacterial toxin-induced cellular dysfunction. Inspired by the fusogenic property of nanodroplets and immunomodulatory functions of metal nanoparticles, biofilm targeted anti-virulent immunomodulatory cationic nanoparticle shelled nanodroplets (C-AgND) is fabricated to completely disintegrate and eradicate the Staphylococcus aureus (S. aureus) biofilm. The specific binding of C-AgND neutralizes the negatively charged EPS layer, causing their destabilization followed by penetration of the nanoformulation into the biofilm matrix, killing the persister cells. Consequently, C-AgND eliminates the virulence property of the S. aureus biofilm through α-hemolysin neutralization. C-AgND promotes a strong immunomodulatory effect by polarizing macrophages into their M1 phenotype to induce phagocytosis of the disintegrated biofilm-released residual cells, rejuvenating the host's innate immune responses for the complete eradication of the biofilm. Moreover, the ex vivo skin wound infection model illustrates an excellent biofilm eradication efficacy of C-AgND in comparison to the commercial ones, rendering them to be a promising replacement of existing antibiofilm agents in clinical application.

Cryptocarya moschata fractions decrease planktonic cells and biofilms of Candida albicans and Streptococcus mutans.

Extracts of Cryptocarya species have been shown to reduce biofilms, demonstrating their antimicrobial effects. The extracts can be fractionated to optimize their potential. In this study, we evaluated the antimicrobial activity of Cryptocarya moschata fractions against planktonic cells and biofilms of Candida albicans and Streptococcus mutans. Four fractions were prepared: 100% hexane, acetate/hexane 1:1, 100% ethyl acetate, and water. The effect of the fractions on planktonic cells was assessed by counting the colony-forming units per milliliter (CFU/mL). Biofilm tests included CFU/mL, cell metabolic activity, and qualitative analysis using confocal laser scanning microscopy (CLSM). Results were analyzed by the Mann-Whitney U test (α = 0.05). The fractions contained lipophilic constituents, styrylpyrones, glycosylated flavonoids, and alkaloids. Acetate/hexane (1:1) and 100% ethyl acetate fractions reduced the CFU/mL of planktonic C. albicans. C. moschata fractions did not affect planktonic S. mutans. For biofilms, the fractions reduced the CFU/mL (from 2-5 logs) and cell metabolic activity (approximately 80% reduction in a single-species biofilm). CLSM showed the fractions reduced microorganism viability and damaged the extracellular matrix of biofilms. We conclude that the acetate/hexane 1:1 and 100% ethyl acetate C. moschata fractions exhibit antimicrobial effects against biofilms.

The Biofilm Removal and Bactericidal Effect of an 810-nm High-Power Laser on an Orthodontic Bracket Surface: An In Vitro Study.

Objective: The present study aimed to analyze the biofilm removal and bactericidal effect of laser treatment alone and laser combined with ultrasonic scaling on orthodontic brackets. It also assessed whether the use of a laser can improve the efficiency of biofilm removal and bactericidal effect compared with traditional ultrasonic instrumentation. Background Data: Streptococcus mutans (S. mutans) can lead to white spots and dental caries. Orthodontic brackets make teeth cleaning more difficult, and biofilms or bacteria on the surface of brackets worsen the oral environment, which may cause some oral diseases. Laser can be used for biofilm removal and killing bacteria on the surface of an object through thermal, photochemical, and pressure effects, which is widely used in the treatment of oral diseases. Methods: A total of 600 mandibular incisor brackets were collected for this study. Among these, 320 unused brackets were used for the S. mutans crystal violet assay (n = 160) and for S. mutans live/dead bacterial staining (n = 160). Another 280 brackets, obtained from patients who had undergone therapy for over two years, were used for the mature multispecies biofilms removal assay (n = 120) and multispecies bacterial live/dead bacterial staining (n = 160). Ultrasonic scaling, laser, and laser combined with ultrasonic scaling were applied to the labial surface of brackets covered by S. mutans biofilm or mature multispecies biofilms. Specifically, we used the following three methods: ultrasonic scaling for 10 sec without laser; 810-nm laser (Doctor Smile, Italy, LA5D0 001.1) with 0.3-mm spot size at total 21.2 kJ/cm2 for 10 sec; and 810-nm laser at total 10.6 kJ/cm2 for 5 sec, followed by ultrasonic scaling for 5 sec. The 810-nm diode laser removed biofilms with a power of 1.5 W and a power density of 2.12 kW/cm2. The S. mutans biofilm was examined using crystal violet assay, and scanning electron microscopy (SEM) was used for mature multispecies biofilms to evaluate the effect of the three methods on biofilm removal. Live/dead bacterial staining was used to examine the bactericidal effect on remaining biofilms by confocal laser scanning microscopy (CLSM). Results: For S. mutans biofilm, the optical density (OD) value and live/dead bacterial ratio in the laser and the laser combined with ultrasonic scaling groups were significantly lower than those in the ultrasonic scaling group (p < 0.05); moreover, the OD value and the live/dead bacterial ratio in laser treatment combined with ultrasonic scaling and laser treatment alone showed no significant difference (p > 0.05). For mature multispecies biofilms, the percentage of biofilm coverage after treatment was higher in the laser group than in the ultrasonic scaling group (p < 0.05) and lower in the laser combined with ultrasonic scaling group than in the ultrasonic scaling group (p < 0.05), and live/dead bacterial staining showed that laser treatment alone killed the most bacteria, followed by laser treatment combined with ultrasonic scaling, while ultrasonic scaling alone seldom killed bacteria. Conclusions: Laser treatment alone has a better bactericidal effect and can also remove more S. mutans biofilm than ultrasonic scaling alone, but it fails to remove more mature multispecies biofilms. Laser treatment combined with ultrasonic scaling can remove more S. mutans biofilm and mature multispecies biofilms than ultrasonic scaling alone and also has a better bactericidal effect than ultrasonic scaling alone on a bracket surface.

Inhibitory Effect of Phenethyl Isothiocyanate on the Adhesion and Biofilm Formation of Staphylococcus aureus and Application on Beef

This study aimed to explore the mechanism by which phenethyl isothiocyanate (PEITC) inhibited the adhesion and biofilm formation of Staphylococcus aureus (S. aureus). PEITC exhibited antimicrobial efficacy against S. aureus, demonstrating a minimum inhibition concentration (MIC) of 1 mmol/L. PEITC exerted its antibacterial effect by disrupting cell membrane integrity, and it decreased total adenosine triphosphate (ATP) production after 1 and 4 h treatment. PEITC at 0.5 mmol/L increased the level of intracellular reactive oxygen species (ROS) by 26.39% compared to control. The mature biofilm of S. aureus was destroyed by 86.4% after treatment with PEITC for 24 h. Adhesion tests revealed that PEITC at 0.5 mmol/L reduced 44.51% of the S. aureus that adhered to NCM460 cells. Furthermore, at the genetic level, PEITC significantly downregulated the related genes by 31.26% to 97.04%, including agrB, agrD, isdA, ebh, luxS, fnbA, and icaR. Moreover, PEITC markedly inhibited S. aureus proliferation in beef preserved at temperatures of 25 and 4 °C, respectively. In summary, the present study suggests that PEITC effectively inhibits the adhesion and biofilm formation of S. aureus by affecting the relevant genes of S. aureus and holds promise for microbial management in meat products.

Therapeutic Potential of Insect Defensin DLP4 Against Staphylococcus hyicus-Infected Piglet Exudative Epidermitis

Background/Objectives: The emergence of resistance to Staphylococcus hyicus (S. hyicus), the major cause of exudative epidermatitis (EE) in piglets, has led to the need for new antimicrobial agents. The study aimed to evaluate the potential efficacy of the insect defensin DLP4 against EE in piglets caused by clinically isolated S. hyicus ACCC 61734. Methods and Results: DLP4 showed strong antibacterial activity against S. hyicus ACCC 61734 (minimum inhibitory concentration, MIC: 0.92 μM, median effect concentration, EC50: 3.158 μM). DLP4 could effectively inhibit the formation of S. hyicus early biofilm with an inhibition rate of 95.10–98.34% and eradicate mature biofilm with a clearance rate of 82.09–86.41%, which was significantly superior to that of ceftriaxone sodium (CRO). Meanwhile, DLP4 could efficiently inhibit bacteria in early and mature biofilm, killing up to 95.3% of bacteria in early biofilm and 87.2–90.3% of bacteria in mature biofilm. The results showed that DLP4 could be effective in alleviating the clinical symptoms of EE by down-regulating the nuclear factor κB (NF-κB) signaling pathway, balancing cytokines, inhibiting bacterial proliferation, and reducing organ tissue damage. Conclusions: This study firstly demonstrated the potential efficacy of DLP4 against EE caused by S. hyicus ACCC 61734 infection in piglets, which may be used as an alternative to antibiotics in treating EE.

Pseudomonas aeruginosa in the Frontline of the Greatest Challenge of Biofilm Infection—Its Tolerance to Antibiotics

P. aeruginosa biofilms are aggregates of bacteria surrounded by a self-produced matrix which binds to some antibiotics such as aminoglycosides. P. aeruginosa biofilms are tolerant to antibiotics. The treatment of biofilm infections leads to a recurrence of symptoms after finishing antibiotic treatment, although the initial clinical response to the treatment is frequently favorable. There is a concentration gradient of oxygen and nutrients from the surface to the center of biofilms. Surface-located bacteria are multiplying and metabolizing, whereas deeper located bacteria are dormant and tolerant to most antibiotics. Colistin kills dormant bacteria, and combination therapy with colistin and antibiotics which kills multiplying bacteria is efficient in vitro. Some antibiotics such as imipenem induce additional production of the biofilm matrix and of chromosomal beta-lactamase in biofilms. Biofilms present a third Pharmacokinetic/Pharmacodynamic (PK/PD) micro-compartment (first: blood, second: tissue, third: biofilm) which must be taken into consideration when calculations try to predict the antibiotic concentrations in biofilms and thereby the probability of target attainment (PTA) for killing the biofilm. Treating biofilms with hyperbaric oxygen to wake up the dormant cells, destruction of the biofilm matrix, and the use of bacteriophage therapy in combination with antibiotics are promising possibilities which have shown proof of concept in in vitro experiments and in animal experiments.

Effect of amikacin-humic acid combination on Acinetobacter baumannii biofilm: an in vitro and in silico study.

Aim: Acinetobacter baumannii (AB) is a clinically important bacterial pathogen responsible for nosocomial infections. The biofilm-forming capability of these pathogens reduces the antibiotic penetration and its efficacy, thereby complicating the treatment. The current work aims to isolate the most potent biofilm-forming Acinetobacter species from clinical isolates of the patient samples and to evaluate the efficacy of the amikacin-humic acid combination against it.Methods: The combination effect of Amikacin-Humic (AMK-HUM) acid against the highest biofilm-producing A. baumannii SLMK001 was studied via in-vitro (microscopic analysis) and in-silico (Network Pharmacology) analysis.Results: The amikacin-humic acid combination significantly inhibited both the biofilm formation and cell viability of A. baumannii SLMK001. The images observed via Scanning Electron Microscope (SEM) showed a significant decrease in the biofilm matrix. Confocal Laser Scanning Microscope (CLSM) confirmed a reduction of the Z value of its three-dimensional structure. Further, the Network Pharmacology approach supported these experimental findings by identifying the key targets of the amikacin-humic acid combination against the biofilm pathways of A. baumannii.Conclusion: The in-vitro results aligned with the in-silico findings, indicating that the AMK-HUM combination is a promising treatment that significantly activates the key proteins against A. baumannii biofilm formation and pathogenesis.

Track of methylparaben in the bulk phase and on the extracellular matrix of dual-species biofilms: Biodegradation and bioaccumulation

Methylparaben (MP) is a preservative considered an environmental contaminant of emerging concern due to its persistence in water sources, including drinking water (DW). This study assesses the interaction between MP and dual-species biofilms of Acinetobacter calcoaceticus and Stenotrophomonas maltophilia. These biofilms were grown under realism-based conditions in a multiple-cylinder biofilm reactor on polypropylene (PPL) surfaces, for 7 days, and then exposed to MP at 0.5 mg/L for three consecutive days. S. maltophilia predominantly succeeds within these biofilms compared to A. calcoaceticus. Exposure to MP resulted in a 4-fold increase in the number of culturable cells and a 1.4-fold rise in polysaccharide content, suggesting that bacterial cells may utilize MP as a carbon source to enhance biofilm fitness. MP was found to adsorb to PPL with biofilms following a pseudo-second-order kinetic model. Circa 37 % of MP adsorbed to PPL after 3 days of exposure. Besides that, MP was biodegraded by biofilms following an apparent first-order kinetic model. Part (25 %) of the MP was biodegraded whereas only 0.02 % bioaccumulated on the biofilm matrix. Biodegradation was related to esterase and lipase activity. The results provide new insights into the interaction between MP with biofilms and materials used in DW industries.

Characterization and genomic analysis of phage vB_SmaP_c9-N, a novel Stenotrophomonas maltophilia podophage with antibiofilm activity.

Stenotrophomonas maltophilia strains are increasingly emerging as multidrug-resistant pathogens. Moreover, S. maltophilia commonly produces biofilms that enhance antibiotic resistance in bacteria. Phages are effective alternative drugs for treating S. maltophilia infections. In this study, the lytic phage vB_SmaP_c9-N (abbreviated as Φc9-N), which is specific for S. maltophilia, was isolated from Nanhu Lake, Wuhan, China. Electron microscopy observation revealed that Φc9-N is a podophage. Φc9-N is stable over a wide pH range, from pH 4 to 10, and its activity did not change after storage at 4°C for 2months. The latency period of Φc9-N is 5 min, and its outbreak period is 35min. Antibacterial tests showed that Φc9-N could effectively inhibit the growth of S. maltophilia c24. Moreover, the biofilm production of S. maltophilia c24 decreased when Φc9-N was administered either to the forming biofilm or to the mature biofilm. These results suggest that Φc9-N has application potential in clinical treatment. The genome of Φc9-N is a dsDNA of 43,170 bp with 55 putative unidirectional genes, 18 of which were assigned putative functions, while other genes encoded hypothetical proteins. Genome sequence comparisons and phylogenetic analysis indicated that Φc9-N represents a new species, and, together with the Stenotrophomonas phages BUCT700, BUCT703, BUCT598, and vB_SmaS_P15, can be included in the newly proposed genus "Maltovirus" in the family Autographiviridae.