Biofilm Viability Research Articles

Integrated Biofilm Dispersion and Virulence Responsiveness for Targeted Treatment of Pseudomonas aeruginosa Infection in Lungs

AbstractThe self‐organization of microbes into biofilms provides multiple benefits including tolerance to mechanical stress and resistance to immune defences and antibiotics. Coupled to a compromised mucociliary function, these traits have dire consequences in cystic fibrosis patients – persistent infections are the main reason for morbidity and mortality. Thereby, disease progression is associated with universal colonization by Pseudomonas aeruginosa, which selects for a slimy phenotype to adapt to the lung microenvironment. Recognizing this, drug‐delivery vehicles that break down the mucoid extracellular matrix made of alginate are designed to enable better penetration and biofilm dispersion. In parallel, a protective layer responds to the proteolytic activity of the pathogen and thus controls drug availability. To realize this architecture, silica nanoparticles are loaded with imipenem, and then coated with elastin and alginate lyase in a layer‐by‐layer fashion using ultrasound. The nanoscale formulations eradicate up to 80% of the total biomass and reduce the bacterial viability in biofilms by 3 logs, considerably outperforming the bulk antibiotic in vitro, whereby the effects are correlated to changes in the viscoelasticity. Furthermore, the stimuli‐responsive nanocarriers are safe and effective in animal models of P. aeruginosa infection, presenting a considerable therapeutic promise in the challenging context of lung diseases.

Innovative Microencapsulation of Polymyxin B for Enhanced Antimicrobial Efficacy via Coated Spray Drying.

This study aims to develop an innovative microencapsulation method for coated Polymyxin B, utilizing various polysaccharides such as hydroxypropyl β-cyclodextrin, alginate, and chitosan, implemented through a three-fluid nozzle (3FN) spray drying process. High-performance liquid chromatography (HPLC) analysis revealed that formulations with a high ratio of sugar cage, hydroxypropyl β-cyclodextrin (HPβCD), and sodium alginate (coded as ALGHCDHPLPM) resulted in a notable 16-fold increase in Polymyxin B recovery compared to chitosan microparticles. Morphological assessments using fluorescence labeling confirmed successful microparticle formation with core/shell structures. Alginate-based formulations exhibited distinct layers, while chitosan formulations showed uniform fluorescence throughout the microparticles. Focused beam reflectance and histograms from fluorescence microscopic measurements provided insights into physical size analysis, indicating consistent sizes of 6.8 ± 1.2 μm. Fourier-transform infrared (FTIR) spectra unveiled hydrogen bonding between Polymyxin B and other components within the microparticle structures. The drug release study showed sodium alginate's sustained release capability, reaching 26 ± 3% compared to 94 ± 3% from the free solution at the 24 h time point. Furthermore, the antimicrobial properties of the prepared microparticles against two Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, were investigated. The influence of various key excipients on the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values was evaluated. Results demonstrated effective bactericidal effects of ALGHCDHPLPM against both E. coli and P. aeruginosa. Additionally, the antibiofilm assay highlighted the potential efficacy of ALGHCDHPLPM against the biofilm viability of E. coli and P. aeruginosa, with concentrations ranging from 3.9 to 500 μg/m. This signifies a significant advancement in antimicrobial drug delivery systems, promising improved precision and efficacy in combating bacterial infections.

Plasma activated water pre-treatment substantially enhances phage activity against Proteus mirabilis biofilms

The ongoing antimicrobial resistance crisis has incentivised research into alternative antibacterial and antibiofilm agents. One of them is plasma-activated water (PAW), which is produced by exposing water to a cold plasma discharge. This process generates a diverse array of reactive oxygen and nitrogen species (ROS/RNS) with antimicrobial properties. Another intensively studied class of alternative antimicrobials are bacteriophages, attracting attention due to their specificity and strong antibacterial activity. As combinations of different types of antimicrobials are known to often exhibit synergistic interactions, in this study we investigated the combined use of cold atmospheric-pressure plasma-activated water and the bacteriophage vB_PmiS_PM-CJR against Proteus mirabilis biofilms as a potential option for treatment of catheter-associated urinary tract infections (CAUTIs).We compared the effect of two cold plasma discharge setups for PAW production on its antimicrobial efficacy against P. mirabilis planktonic and biofilm cultures. Next, we assessed the stability of the phage vB_PmiS_PM-CJR in PAW. Finally, we tested the antimicrobial activity of the phages and PAW against biofilms, both individually and in combinations.Our findings demonstrate that the combination of PAW with phage is more effective against biofilms compared to individual treatments, being able to reduce the number of biofilm-embedded cells by approximately 4 log. We were also able to show that the order of treatment plays an important role in the anti-biofilm activity of the phage-PAW combination, as the exposure of the biofilm to PAW prior to phage administration results in a stronger effect than the reverse order.This research underlines PAW's ability to potentiate phage activity, showcasing a considerable reduction in biofilm viability and biomass. Additionally, it contributes to the growing body of evidence supporting the use of phage-based combinatorial treatments. Overall, this sequential treatment strategy demonstrates the potential of leveraging multiple approaches to address the mounting challenge of antibiotic resistance and offers a promising avenue for enhancing the efficacy of CAUTI management.

Kanamycin promotes biofilm viability of MRSA strains showing extremely high resistance to kanamycin

Staphylococcus aureus is widely distributed in environment and can cause various human infection and food poisoning cases. Also, this pathogen is a typical biofilm former, which further complicates its pathogenicity. Antibiotics have been widely used to eliminate pathogenic bacteria, but their indiscriminate use has also led to the widespread emergence of drug-resistant bacteria, such as Methicillin-Resistant Staphylococcus aureus (MRSA). In this study, the effect of antibiotics on biofilm formation of MRSA strains 875 and 184 was explored. Firstly, MRSA 875 belongs to SCCmec type IV, ST239, carrying the atl, icaA, icaD, icaBC, and aap genes, and MRSA 184 belongs to SCCmec type II, ST5, carrying the atl, icaD, icaBC, aap, and agr genes. Then, a total of 8 antibiotics have been selected, including kanamycin, gentamycin, cipprofloxacin, erythromycin, meropenem, penicillin G, tetracycline, vancomycin. Minimum inhibitory concentrations (MICs) of each antibiotic were determined, and MIC of MRSA 875 and 184 to kanamycin/gentamicin are 2048/64 μg/mL and 2048/4 μg/mL, respectively. A total of 10 concentrations, ranging from 1/128 to 4 MIC with 2-fold, were used to study biofilm formation. Biofilm biomass and viability were determined during different phases, including initial adhesion (8 h), proliferation (16 h), accumulation (24 h) and maturation (48 h). Importantly, kanamycin at specific concentrations showed significant promotion of biofilm biomass and biofilm viability, with none of such observation acquired from other antibiotics. This study provides scientific basis and new research ideas for the quality control technology of microorganisms and safety prevention of MRSA.

Enhancing Removable Partial Dentures Hygiene: Investigating Mucolytic Agents and Biocides for Disrupting Biofilms and Improving Antimicrobial Efficacy.

This study evaluates the antibiofilm action of 2.5 mg/mL peracetic acid (PA), 0.5 mg/mL cetylpyridinium chloride (CPC), and 160 mg/mL N-Acetylcysteine (NAC) against multispecies biofilm of Streptococcus mutans, Staphylococcus aureus, Candida albicans, and Candida glabrata, developed on surfaces of heat-polymerizing acrylic resin (AR) and cobaltchromium (Co-Cr) alloy. A multispecies biofilm was grown on the surface of AR and Co-Cr specimens (Ø 12×3mm). After biofilm maturation, the specimens were immersed in experimental solutions and evaluated through biofilm viability (CFU) (n&#61;9), biofilm metabolic activity (XTT) (n&#61;9), biofilm-covered areas (Live/Dead) (n&#61;2), effects on the extracellular polymeric substance (EPS) (n&#61;2) and biofilm morphology (n&#61;1). Data were analyzed by ANOVA and the Tukey post-test or Kruskal-Wallis followed by the Dunn post-test (α&#61;.05). Overall, all evaluated solutions impacted biofilm viability. PA presented wider activity by reducing CFU of all microorganisms on both surfaces, XTT (P<.001) and Live/Dead (P<.001). NAC had a notorious effect in reducing the viability of bacteria without affecting the yeasts. NAC reduced XTT on AR (P&#61;.006) and Co-Cr (P&#61;.003) but did not reduce the aggregated biofilm layer. CPC had distinct effect according to the surface, being most effective in reducing CFU on AR than the Co-Cr surface. However, it did not influence XTT, and the amount of residual aggregated biofilm. PA provided the greatest antibiofilm action, while CPC and NAC showed intermediate action. Nonetheless, no solution was able to completely remove the biofilm adhered to the surfaces of heat-polymerizing AR and Co-Cr alloy.

A pH-modulated polymeric drug-release system simultaneously combats biofilms and planktonic bacteria to eliminate bacterial biofilm

Bacterial infection, leading to biofilm formation, raises grave concerns. With the aim of eliminating both integrity of biofilms and individual bacterial cells, it is desirable to develop bacterial biofilm-eliminating material to be combined with dispersal and anti-microbial agents. Herein, a biofilm-eliminating enzyme (α amylase) was incorporated in novel pH-sensitive polymer hydrogel, Poly (methacrylic acid-co-2-(dimethylamino)ethyl methacrylate), which encapsulates drug-loaded porous Zeolitic Imidazolate Framework-8 (ZIF-8). At body temperature (37 °C) the maximum swelling of the hydrogel was observed at pH 8.5, which assisted in rupturing biofilm by releasing α amylase. Amylase, encapsulated in polymer matrix reached 90% release after 6 days at 8.5, while CIP was released 96% on day 23 at pH 8.5. The antibiofilm activity of drug-releasing system was assessed against E. coli and S. aureus. The overall bacterial viability of biofilms showed significant time-dependent declines, when exposed to drug-loaded hydrogel. Importantly, cytotoxicity experiments showed excellent biocompatibility of hydrogel. In the in vivo deep wound infection model, drug-loaded hydrogel treatment showed complete wound closure with proper tissue regeneration. No inflammatory, or systemic toxicity of hydrogel was observed. The drug release from hydrogel is initiated only upon receiving physiological cue arising due to biofilm formation. Therefore, this hydrogel serves as reservoir for antibacterial drug, which release it only in presence of biofilm and thus work for sustained release for long period, compared to conventional drug loaded films or coatings. A powerful modality for management of biofilm-associated infections can be provided by the proposed synergistic strategy for complete elimination of biofilms.

Characterization of Two Novel Endolysins from Bacteriophage PEF1 and Evaluation of Their Combined Effects on the Control of Enterococcus faecalis Planktonic and Biofilm Cells.

Endolysin, a bacteriophage-derived lytic enzyme, has emerged as a promising alternative antimicrobial agent against rising multidrug-resistant bacterial infections. Two novel endolysins LysPEF1-1 and LysPEF1-2 derived from Enterococcus phage PEF1 were cloned and overexpressed in Escherichia coli to test their antimicrobial efficacy against multidrug-resistant E. faecalis strains and their biofilms. LysPEF1-1 comprises an enzymatically active domain and a cell-wall-binding domain originating from the NLPC-P60 and SH3 superfamilies, while LysPEF1-2 contains a putative peptidoglycan recognition domain that belongs to the PGRP superfamily. LysPEF1-1 was active against 89.86% (62/69) of Enterococcus spp. tested, displaying a wider antibacterial spectrum than phage PEF1. Moreover, two endolysins demonstrated lytic activity against additional gram-positive and gram-negative species pretreated with chloroform. LysPEF1-1 showed higher activity against multidrug-resistant E. faecalis strain E5 than LysPEF1-2. The combination of two endolysins effectively reduced planktonic cells of E5 in broth and was more efficient at inhibiting biofilm formation and removing biofilm cells of E. faecalis JCM 7783T than used individually. Especially at 4 °C, they reduced viable biofilm cells by 4.5 log after 2 h of treatment on glass slide surfaces. The results suggest that two novel endolysins could be alternative antimicrobial agents for controlling E. faecalis infections.

Inhibitory effect of helium cold atmospheric plasma on cariogenic biofilms

ABSTRACT This study aimed to determine the effects of low-temperature plasma jet produced in gas helium (LTP-helium) on cariogenic biofilms composedby Streptococcusmutans, Streptococcus sanguinis and Streptococcus gordonii, and also by the combination of Candida albicans, Lactobacillus acidophilus and S. mutans. Biofilms were treated for 1, 3, 5, and 7 minutes. A 0.12% chlorhexidine solution was used as the positive control and sterile physiologic solution was the negative control. Biofilm viability was analyzed by viable cell recovery, scanning electron microscopy, and confocal laser scanning microscopy. All assays were performed intriplicate in three independent experiments. Multispecies biofilms exposed to LTP-helium had a significant reduction in viability when compared to the negative control (p < 0.0001). For biofilm formedby S. mutans, S. sanguinis, and S. gordonii, LTP treatments for 5 and 7 minutes caused similar reduction of morethan 2 log10. Also, a significant reduction in the viability of biofilms formedby C. albicans, L. acidophilus, and S. mutans was detected (p < 0.0001). In conclusion, LTP-helium reduced theviability of cariogenic biofilms with different microbial compositions, which indicates that LTP-helium is a potential tool for developing new protocols for dental caries prevention and treatment.

Exploring the efficacy of in-vitro low-temperature plasma treatment on single and multispecies dental cariogenic biofilms

The primary aim of this study was to investigate the impact of treatment with low-temperature plasma (LTP) for varying exposure durations on a multispecies cariogenic biofilm comprising C. albicans, L. casei, and S. mutans, as well as on single-species biofilms of L. casei and C. albicans, cultured on hydroxyapatite discs. Biofilms were treated with LTP-argon at a 10 mm distance for 30 s, 60 s, and 120 s. Chlorhexidine solution (0.12%) and NaCl (0.89%) were used as positive (PC) and negative controls (NC), respectively. Argon flow only was also used as gas flow control (F). Colony-forming units (CFU) recovery and confocal laser scanning microscopy (CLSM) were used to analyze biofilm viability. LTP starting at 30 s of application significantly reduced the viability of multispecies biofilms by more than 2 log10 in all treated samples (p < 0.0001). For single-species biofilms, L. casei showed a significant reduction compared to PC and NC of over 1 log10 at all exposure times (p < 0.0001). In the case of C. albicans biofilms, LTP treatment compared to PC and NC resulted in a significant decrease in bacterial counts when applied for 60 and 120 s (1.55 and 1.90 log10 CFU/mL, respectively) (p < 0.0001). A significant effect (p ≤ 0.05) of LTP in single-species biofilms was observed to start at 60 s of LTP application compared to F, suggesting a time-dependent effect of LTP for the single-species biofilms of C. albicans and L. casei. LTP is a potential mechanism in treating dental caries by being an effective anti-biofilm therapy of both single and multispecies cariogenic biofilms.

The effectiveness of antimicrobial photodynamic therapy on catheter infection model

Background/AimThis experimental study aimed to examine the effectiveness of transdermal antimicrobial photodynamic therapy (APDT) with and without antimicrobial lock therapy (ALT), on catheter biofilms. MethodsS. epidermidis and C. orthopsilosis biofilms were formed within peripheral venous catheters positioned in the marginal ear veins of New Zealand white rabbits. Biofilm formation was confirmed with scanning electron microscopy in two catheters. 24 catheters with staphylococcal biofilms and 24 with fungal biofilms were treated with APDT, ALT or “APDT plus ALT” for five days. Six catheters were separated as controls. APDT was applied with a red colored LED lamp and methylene blue as the photosensitizer. Vancomycin lock solutions were used as ALT for staphylococcal biofilms and amphotericin B for fungal biofilms. The effect of treatment procedures was evaluated by intraluminal biofilm viability testing based on spectrophotometric evaluation, and a quantitative (OD) value was obtained for each catheter. ResultsThe mean OD values obtained by 600 nm spectrophotometric reading at 24 h (biofilm viability) after “ALT”, “APDT” and “ALT plus APDT” procedures were 0.363, 0.151 and 0.128 for S. epidermidis and 0.092, 0.104 and 0.227 for C. orthopsilosis, respectively. All these OD values obtained after treatment procedures were lower than controls for both S. epidermidis (OD: 0,802) and C. orthopsilosis (OD: 0,315), although there were large fluctuations in our results. ConclusionsOur results suggest that transdermal APDT may be an effective method for treating staphylococcal and candida biofilms formed within intravenous catheters in our rabbit ear model. The combined use of APDT and ALT might be beneficial in these staphylococcal biofilms.

Anti-biofilm properties of Portuguese honeys against multi-drug resistant microorganisms: A promising strategy for chronic wounds healing

Honey is considered a promising strategy for chronic wound management, being a possible solution for bacterial resistance problem mostly due to its physical-chemical features. However, these properties might vary significantly depending on the honey floral source. Dark-colored honey, such as chestnut honey, is related to increased antioxidant and bactericidal properties. This study aims to assess biofilms of multidrug-resistant microorganisms in honey samples from the Natural Park of Montesinho, in Portugal, which is considered a region with predominant chestnut floral source. The minimum inhibition concentration (MIC) of honey was tested for Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida albicans. The biofilm removal as well as the metabolic inactivation of the biofilms were evaluated using three honey concentrations: 1xMIC, 5xMIC, and 10xMIC. The biofilm viability was analyzed with fluorescent microscopy. Honey samples were characterized according to the pollen profile, phenolic composition, H2O2 levels, tyrosinase inhibition, and pH. The antioxidant capacity was determined through FRAP and ABTS assays. Most of the honey samples exhibited a MIC of 6.75%. Biofilm removal efficacy differs among samples. Regression analysis evidenced a positive correlation between the biofilm removal and concentrations of H2O2, vanillic acid, pinocembrin, Cytisus striatus and Corrigiola telephiifolia, and pH. The honey concentration of 10xMIC was the most effective to inhibit the biofilm metabolism and viability. The concentrations of C. sativa, H2O2, and the anti-tyrosinase and FRAP activities were also positively correlated. The honey samples had a dominant presence of Castanea sativa pollen and were effective against biofilms formed by multidrug-resistant microorganisms.

Bacteriophage Therapy on an In Vitro Wound Model and Synergistic Effects in Combination with Beta-Lactam Antibiotics.

One of the primary opportunistic pathogens that can cause a wide range of diseases is Pseudomonas aeruginosa. This microorganism can become resistant to practically every antibacterial currently in use, including beta-lactam antibiotics. Its ability to proliferate as biofilm has been linked to, among other things, the failure of antimicrobial therapies. Due to a variety of virulence factors and host immune system modifications, P. aeruginosa is one of the most significant and common bacteria that colonize wounds and burns. A novel therapeutic option for treating these multidrug-resistant (MDR) bacterial infections is the combination of antibiotics and bacteriophages. This approach has been linked to improved biofilm penetration, a decreased selection of antibiotic and bacteriophage resistance, and an enhanced antibacterial impact. Combining the F1Pa bacteriophage and beta-lactam antibiotics reduced the viability of the mature biofilm of MDR P. aeruginosa strains and suppressed bacterial growth in vitro. F1Pa critically reduced the amount of biofilm that MDR P. aeruginosa clinical strains formed in the in vitro wound model. These findings highlight the bacteriophage F1Pa's therapeutic potential as a prophylactic topical treatment against MDR pseudomonal infections in wounds and burns.

Preventing proximal enamel caries in neighboring tooth with glass ionomer cement restoration and silver diamine fluoride pretreatment

ObjectiveTo investigate caries preventive effects of 38 % silver diamine fluoride (SDF) pretreatment on neighboring tooth proximal to glass ionomer cement (GIC), including conventional GIC (CGIC) and resin-modified GIC (RMGIC) restorations in an in vitro model. Methods: Human tooth blocks were restored withSDF+CGIC (Group 1), CGIC (Group 2), SDF+RMGIC (Group 3) or RMGIC (Group 4). Enamel specimen simulating proximal surface of neighboring tooth was placed in proximity to the restorations. The specimen underwent cariogenic challenge with cross-kingdom biofilm of Streptococcus mutans, Lacticaseibacillus casei and Candida albicans. After cariogenic challenge, the biofilm's growth kinetics, viability, and morphology were evaluated by propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR), confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), respectively. The enamel lesion depth, surface morphology and crystal characteristics were determined by micro-computed tomography (micro-CT), SEM and X-ray diffraction (XRD), respectively. ResultsPMA-qPCR demonstrated lower microbial growth in Group 1 and 3 compared with Group 2 and 4 (p < 0.05). CLSM showed the dead−to-live ratio in Groups 1–4 were 1.15±0.12, 0.53±0.13, 1.10±0.24 and 0.63±0.10, respectively (Group 1,3 > 2,4, p < 0.05). SEM revealed Groups 1 and 3 had scattered biofilm whereas Group 2 and 4 had confluent biofilm. Micro-CT showed the enamel lesion depths (µm) were 98±9, 126±7, 103±6 and 128±7 for Group 1 to 4, respectively (Group 1,3 < 2,4, p < 0.05). SEM revealed oriented and ordered enamel prismatic patterns in Group 1 and 3, not in Group 2 and 4. XRD showed the reflections of hydroxyapatite in Groups 1 and 3 were sharper than Groups 2 and 4. ConclusionSDF pretreatment enhances the preventive effect of GIC on proximal enamel surface on neighboring tooth through inhibiting cariogenic biofilm, reducing enamel demineralization and promoting enamel remineralization. Clinical significanceSDF pretreatment of GIC restorations can help prevent caries on neighboring teeth, particular for patients with high caries risk.

Antifungal effect of a metabolite of Pseudomonas aeruginosa LV strain on azole-resistant Candida albicans

Candida albicans remains the most common agent of candidiasis worldwide. This yeast is generally sensitive to most antifungals, however, the emergence of azole-resistant C. albicans has been reported. In addition, this microorganism can form biofilms on various surfaces, making it difficult to treat infections. In this study, the effect of secondary metabolites of Pseudomonas aeruginosa strain LV on planktonic and sessile cells of C. albicans, with different genotypes and susceptibility profile to fluconazole and voriconazole, was evaluated. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the semi-purified fraction F4a ranged from 1.56 to 6.25 μg/mL and 6.25 to 25 μg/mL, respectively. Fluopsin C appears to be the antifungal component of F4a. The semi-purified fraction and fluopsin C showed fungicidal activity, dose and time dependent. F4a caused severe damage to the morphology and ultrastructure of planktonic fungal cells, and significantly reduced the viability of 24-hour biofilms, with MIC for sessile cells from 12.5 to 25.0 μg/mL. However, cytotoxicity was detected in mammalian cells for F4a and fluopsin C at concentrations that showed antifungal activity. These results indicate that fluopsin C may be a prototype for the development of new antifungals for C. albicans.

Geraniol Potentiates the Effect of Fluconazole against Planktonic and Sessile Cells of Azole-Resistant Candida tropicalis: In Vitro and In Vivo Analyses.

Candida tropicalis is regarded as an opportunistic pathogen, causing diseases ranging from superficial infections to life-threatening disseminated infections. The ability of this yeast to form biofilms and develop resistance to antifungals represents a significant therapeutic challenge. Herein, the effect of geraniol (GER), alone and combined with fluconazole (FLZ), was evaluated in the planktonic and sessile cells of azole-resistant C. tropicalis. GER showed a time-dependent fungicidal effect on the planktonic cells, impairing the cell membrane integrity. Additionally, GER inhibited the rhodamine 6G efflux, and the molecular docking analyzes supported the binding affinity of GER to the C. tropicalis Cdr1 protein. GER exhibited a synergism with FLZ against the planktonic and sessile cells, inhibiting the adhesion of the yeast cells and the viability of the 48-h biofilms formed on abiotic surfaces. C. tropicalis biofilms treated with GER, alone or combined with FLZ, displayed morphological and ultrastructural alterations, including a decrease in the stacking layers and the presence of wilted cells. Moreover, neither GER alone nor combined with FLZ caused toxicity, and both treatments prolonged the survival of the Galleria mellonella larvae infected with azole-resistant C. tropicalis. These findings indicate that the combination of GER and FLZ may be a promising strategy to control azole-resistant C. tropicalis infections.

Revealing the Viable Microbial Community of Biofilm in a Sewage Treatment System Using Propidium Monoazide Combined with Real-Time PCR and Metagenomics.

Microbial community composition, function, and viability are important for biofilm-based sewage treatment technologies. Most studies of microbial communities mainly rely on the total deoxyribonucleic acid (DNA) extracted from the biofilm. However, nucleotide materials released from dead microorganisms may interfere with the analysis of viable microorganisms and their metabolic potential. In this study, we developed a protocol to assess viability as well as viable community composition and function in biofilm in a sewage treatment system using propidium monoazide (PMA) coupled with real-time quantitative polymerase chain reaction (qPCR) and metagenomic technology. The optimal removal of PMA from non-viable cells was achieved by a PMA concentration of 4 μM, incubation in darkness for 5 min, and exposure for 5 min. Simultaneously, the detection limit can reach a viable bacteria proportion of 1%, within the detection concentration range of 102-108 CFU/mL (colony forming unit/mL), showing its effectiveness in removing interference from dead cells. Under the optimal conditions, the result of PMA-metagenomic sequencing revealed that 6.72% to 8.18% of non-viable microorganisms were influenced and the composition and relative abundance of the dominant genera were changed. Overall, this study established a fast, sensitive, and highly specific biofilm viability detection method, which could provide technical support for accurately deciphering the structural composition and function of viable microbial communities in sewage treatment biofilms.

Synergic Effect of the Antimicrobial Peptide ToAP2 and Fluconazole on Candida albicans Biofilms.

Candida albicans is one of the agents of invasive candidiasis, a life-threatening disease strongly associated with hospitalization, particularly among patients in intensive care units with central venous catheters. This study aimed to evaluate the synergistic activity of the antifungal peptide ToAP2 combined with fluconazole against C. albicans biofilms grown on various materials. We tested combinations of different concentrations of the peptide ToAP2 with fluconazole on C. albicans biofilms. These biofilms were generated on 96-well plates, intravenous catheters, and infusion tubes in RPMI medium at two maturation stages. Scanning electron microscopy and atomic force microscopy were employed to assess the biofilm structure. We also evaluated the expression of genes previously proven to be involved in C. albicans biofilm formation in planktonic and biofilm cells after treatment with the peptide ToAP2 using qPCR. ToAP2 demonstrated a synergistic effect with fluconazole at concentrations up to 25 µM during both the early and mature stages of biofilm formation in 96-well plates and on medical devices. Combinations of 50, 25, and 12.5 µM of ToAP2 with 52 µM of fluconazole significantly reduced the biofilm viability compared to individual treatments and untreated controls. These results were supported by substantial structural changes in the biofilms observed through both scanning and atomic force microscopy. The gene expression analysis of C. albicans cells treated with 25 µM of ToAP2 revealed a decrease in the expression of genes associated with membrane synthesis, along with an increase in the expression of genes involved in efflux pumps, adhesins, and filamentation. Our results highlight the efficacy of the combined ToAP2 and fluconazole treatment against C. albicans biofilms. This combination not only shows therapeutic potential but also suggests its utility in developing preventive biofilm tools for intravenous catheters.

Effect of Novel and Traditional Intracanal Medicaments on Biofilm Viability and Composition

IntroductionThe aim of this study was to test the hypothesis that a combination of D-amino acids (DAAs) and trans-cinnamaldehyde (TC) demonstrates superior antibiofilm activity to calcium hydroxide (CH) and untreated controls. MethodsIn this 3-part in vitro study, the concentration of DAAs (D-methionine, D-leucine, D-tyrosine, and D-tryptophan) that would significantly decrease Enterococcus faecalis and Actinomyces naeslundii biofilm biomass was first determined. Then, the effect of TC + selected DAAs on polymicrobial biofilms was characterized by quantifying the biomass and biofilm viability. Finally, the antibiofilm effects of TC + DAA was compared with CH and untreated controls by (i) determining bacterial viability and (ii) quantifying biofilm matrix composition using selective fluorescence-binding analysis. Statistical analysis was performed using one-way ANOVA and appropriate multiple comparisons test, with P < .05 considered as statistically significant. ResultsTC (0.06%) + D-tyrosine (1 mM) + D-tryptophan (25 mM) significantly reduced the biomass and biofilm viability compared to the control (P < .05). While no significant difference was observed between TC + DAA and CH in the cultivable bacterial counts (P > .05), confocal microscopy demonstrated a significantly greater percentage of dead bacteria in TC + DAA-treated biofilms compared to CH and the control (P < .05). TC + DAA significantly decreased the biovolume and all the examined components of the biofilm matrix quantity compared to the control, while CH significantly reduced only the exopolysaccharide quantity (P < .05). ConclusionThe combination of TC + D-tyrosine + D-tryptophan demonstrated superior antibiofilm activity (biofilm bacterial killing and reduction of matrix quantity) to CH and has potential to be developed as an intracanal medicament.

The combined effect of pH and NaCl on the susceptibility of Listeria monocytogenes to rhamnolipids

The use of natural and sustainable additives, that are less aggressive to the environment, is a trend in the food industry. Rhamnolipids (RL) biosurfactants have shown potential for controlling food pathogens however, due to the presence of free carboxyl groups, the pH and ionic strength may influence the properties of such surfactants. In this study, we describe the antimicrobial activity of RL under different pH values and NaCl concentrations, towards both planktonic and biofilms of Listeria monocytogenes. RL were effective at pH 5.0 and the addition of 5 % NaCl improved the bactericidal efficacy for planktonic and sessile cells. The effect of NaCl was more pronounced at pH above 6 showing a significant increase in RL antimicrobial activity. At pH 7.0 planktonic population was eradicated by RL only when salt was present whereas biofilm viability was decreased by 5 log with MBIC varying from > 2500.0 mg/L (RL) to 39.0 mg/L (RL + 5 % NaCl). Larger vesicular and lamellar RL self-assembly structures were predominant when NaCl was present, suggesting their association with the antimicrobial activity observed. The pH and ionic strength of the medium are important parameters to be considered for the development of RL-based strategies to control L. monocytogenes.

Cold atmospheric plasma inactivates Aspergillus flavus and Fusarium keratoplasticum biofilms and conidia in vitro.

Introduction. Aspergillus flavus and Fusarium keratoplasticum are common causative pathogens of fungal keratitis (FK), a severe corneal disease associated with significant morbidity and vision loss. Escalating incidence of antifungal resistance to available antifungal drugs poses a major challenge to FK treatment. Cold atmospheric plasma (CAP) is a pioneering nonpharmacologic antimicrobial intervention that has demonstrated potential as a broad-spectrum antifungal treatment.Gap statement. Previous research highlights biofilm-associated resistance as a critical barrier to effective FK treatment. Although CAP has shown promise against various fungal infections, its efficacy against biofilm and conidial forms of FK pathogens remains inadequately explored.Aim. This study aims to investigate the antifungal efficacy of CAP against clinical fungal keratitis isolates of A. flavus and F. keratoplasticum in vitro.Methodology. Power parameters (22-27 kVpp, 300-400 Hz and 20-80 mA) of a dielectric barrier discharge CAP device were optimized for inactivation of A. flavus biofilms. Optimal applied voltage and total current were applied to F. keratoplasticum biofilms and conidial suspensions of A. flavus and F. keratoplasticum. The antifungal effect of CAP treatment was investigated by evaluating fungal viability through means of metabolic activity, c.f.u. enumeration (c.f.u. ml-1) and biofilm formation.Results. For both fungal species, CAP exhibited strong time-dependent inactivation, achieving greater than 80 % reduction in metabolic activity and c.f.u. ml-1 within 300 s or less, and complete inhibition after 600 s of treatment.Conclusion. Our findings indicate that CAP is a promising broad-spectrum antifungal intervention. CAP treatment effectively reduces fungal viability in both biofilm and conidial suspension cultures of A. flavus and F. keratoplasticum, suggesting its potential as an alternative treatment strategy for fungal keratitis.