Biofilm Mass Research Articles

Oral ciprofloxacin biofilm activity in a catheter-associated urinary tract infection model.

Catheter-associated urinary tract infections (CA-UTIs) are a common hospital-acquired infection. We examined ciprofloxacin activity in a novel CA-UTI in vitro model. Three ATCC strains [Escherichia coli (ECO)-25922, Klebsiella pneumoniae (KPN)-700721, Pseudomonas aeruginosa (PAE)-27853] and 45 clinical urinary isolates were assessed. Biofilm mass and planktonic bacterial density were quantified during drug-free incubation (72 h) and following ciprofloxacin exposure (equivalent 750 mg orally q12h, 3 days). ECO produced smaller biofilms (6.3 ± 1.1 log10 cfu/cm2) compared with KPN (7.1 ± 0.7 log10 cfu/cm2) and PAE (7.0 ± 1.2 log10 cfu/cm2), which extended along the entire catheter length. Following ciprofloxacin, all isolates with MIC > 4 mg/L had minimal biofilm disruption or planktonic kill. Ciprofloxacin resistance was most common in PAE isolates (10/16 isolates), compared with ECO (3/16 isolates) and KPN (6/16 isolates). Greater ciprofloxacin exposure (AUC0-24/MIC) was required for a 3 log10 biofilm kill for KPN (5858; R2 = 0.7774) compared with ECO (2117; R2 = 0.7907) and PAE (2485; R2 = 0.8260). Due to persistent growth in the bladder, ECO required greater ciprofloxacin exposure for a 3 log10 planktonic kill (5920; R2 = 0.8440) compared with KPN (2825; R2 = 0.9121) and PAE (1760; R2 = 0.8781). Monte Carlo simulation supported a 95% PTA for both a 3 log10 biofilm and planktonic kill for ECO and KPN isolates with MIC ≤ 0.5 mg/L and PAE isolates with MIC ≤ 1 mg/L. In a novel CA-UTI model, following simulated ciprofloxacin therapy, KPN biofilms were comparatively more difficult to disrupt, ECO planktonic growth frequently persisted in the bladder, and PAE had greater propensity for emergence of ciprofloxacin resistance.

Mechanistic Insight into the Anti-Bacterial/Anti-Biofilm Effects of Low Chlorhexidine Concentrations on Enterococcus faecalis-In Vitro Study.

Endodontic treatment failures are often linked to the persistence of Enterococcus faecalis in the root canal system. This study aimed to investigate the antibacterial/antibiofilm mechanism of chlorhexidine (CHX), particularly at low concentrations, against E. faecalis, to improve endodontic treatment protocols. The antibacterial activity of CHX (0.125-20 μg/mL) was evaluated against E. faecalis ATCC 29212 using various assays, including planktonic growth inhibition, colony-forming units (CFUs), membrane permeability and potential assays, high-resolution scanning electron microscopy (HR-SEM), confocal laser scanning microscopy of biofilms, biomass and metabolic activity assays on matured biofilm, and quantitative real-time PCR for gene expression. Statistical analysis was performed using Student's t-test and ANOVA. CHX demonstrated concentration-dependent inhibition of E. faecalis, significantly reducing planktonic growth and CFUs. Membrane assays showed increased permeability and depolarization, indicating damage. HR-SEM revealed morphological changes, such as pore formation, while confocal microscopy showed a reduction in biofilm mass and extracellular substances. Gene expression analysis indicated the downregulation of virulence genes and upregulation of stress response genes. CHX at low concentrations disrupts E. faecalis at multiple levels, from membrane disruption to gene expression modulation, affecting mature biofilm. These findings support the refinement of endodontic disinfection protocols to reduce microbial persistence.

Doxifluridine effectively kills antibiotic-resistant Staphylococcus aureus in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality globally, often exacerbated by infections such as methicillin-resistant Staphylococcus aureus (MRSA). The rise in antibiotic-resistant strains complicates treatment and underscores the need for novel therapeutic drugs. In this paper, we further investigated the antimicrobial potential of a fluoropyrimidine anticancer drug doxifluridine against multidrug-resistant S. aureus. Determination of minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC), monitoring of growth curve, time-kill assays, biofilm bactericidal assays, and chequerboard studies were conducted to evaluate the antibacterial efficacy of doxifluridine. Safety was assessed via hemolysis and cytotoxicity assays, and an in vivo Galleria mellonella larvae model was employed to test protective effects. Doxifluridine demonstrated significant antibacterial activity against clinical multidrug resistance (MDR) S. aureus isolates, with MIC and MBC values ranging from 0.5 to 2 µg/mL and 1 to 4 µg/mL, respectively. The results revealed doxifluridine's potent bactericidal effects within 8 hours. Moreover, doxifluridine-treated bacteria showed a substantial reduction in biofilm mass and viability. Furthermore, chequerboard assays indicated synergistic interactions between doxifluridine and other antibiotics, reducing MIC values by two- to eightfold. More importantly, safety evaluations confirmed that doxifluridine did not exhibit hemolytic toxicity or cytotoxicity. Finally, doxifluridine significantly increased the survival rate of MRSA-infected G. mellonella larvae in vivo. In brief, doxifluridine exhibited promising in vitro and in vivo antibacterial activity against MRSA, suggesting its potential as a repurposed drug for treating resistant bacterial infections in COPD patients.IMPORTANCEThe study provides robust evidence for the antibacterial efficacy of doxifluridine against Methicillin-resistant Staphylococcus aureus in chronic obstructive pulmonary disease (COPD) patients. Its rapid action, ability to disrupt biofilms, and synergistic effects with other antibiotics, combined with a favorable safety profile, highlight its potential as a repurposed therapeutic agent. Future clinical trials will be essential to confirm these findings and pave the way for its integration into clinical practice. This work not only provides candidate for tackling the management of bacterial infections in COPD but also exemplifies the potential of drug repurposing in combating antibiotic-resistant infections.

In-vitro and In-silico evaluation of antimicrobial and antibiofilm effect of Neem oil and Calcium hydroxide nanoparticles against Mutans Streptococci and Enterococcus faecalis isolated from endodontic infections

Different Streptococcal species including Streptococcus mutans, Streptococcus sobrinus and Enterococcus faecalis are commonly isolated in root canal infections including refractory, recurrent, and persistent cases. Calcium hydroxide (Ca (OH)2) has been widely used in endodontics as an intracanal medicament. However, using new antimicrobial herbal alternatives offers promising potentials which can be additionally enhanced by using nanoparticles (NPs). In this study, we evaluate the antimicrobial efficacy and antibiofilm effect of Neem oil including its NPs preparations and we compare the effect of conventional Ca (OH)2 to Ca (OH)2 NPs using standard disc diffusion method and quantitative microtitre dish biofilm formation assay against common pathogens isolated from root canal samples. Molecular docking was used to test the binding of 10 Streptococcal macromolecules to 5 candidate neem active constituents. Neem NPs 0.125 mg/ml showed better antibacterial effect than both Neem 15 mg/ml and Neem 0.15 mg/ml. Ca (OH)2 NPs 0.125 mg/ml also showed better antibacterial effect than each of Ca (OH)2 10 mg/ml and Ca (OH)2 0.1 mg/ml. Best biofilm mass inhibition was achieved by Neem oil 0.15 mg/ml at 74.55% ( IQ: 67.36–87.65) and Neem NPs 0.0125 mg/ml at 59.33% (IQ: 51-–75.27). For Ca (OH)2, the best biofilm mass inhibition was observed with Ca (OH)2 NPs 0.125 mg/ml at 54.7% (IQ: 42.37– 77.25). Both neem oil and neem NPs show promising antibacterial and antibiofilm potential against Mutans Streptococci group at low concentrations and hence are good candidates for use as endodontic medications. In silico analysis shows that both Sitosterol and Gedunin appear to be important active constituents of neem and possible drug candidates. Additionally, Ca (OH)2 NPs showed significantly higher antimicrobial effect against Mutans streptococci group than conventional Ca (OH)2 preparations.

The treatment of Enterococcus faecalis related root canal biofilms with phage therapy

As one of the global concerns, antimicrobial resistance crisis increases the clinical importance of Enterococcus species. Enterococcus faecalis (E. faecalis) specifically penetrates the dentinal tubules and remains prevalent even after endodontic treatment. It has also biofilm forming character as well as the development of resistance to antibiotics. Sodium hypochlorite (NaOCl) is considered the gold standard among antibacterial washing solutions. However, due to its toxic effects, its usage limitations have led to the search for natural, non-toxic alternatives. Phages can be considered an important alternative because of their effects on specific bacteria. The objective of this study was to compare the effect of isolated active vB_Ef1 phage on the removal of E. faecalis biofilm in dentin, together and separately with the chemical irrigation solution NaOCl. As a result of study, the optimal NaOCl solution concentration to be applied with vB_Ef1 phage is 0.5 %, and the use of solution at this value reduces the biofilm mass by 84 %, reaching the highest biofilm mass reduction value. It was found that the combination of phage and NaOCl at appropriate concentrations had the strongest biofilm disruption effect.

Role of the SaeRS Two-Component Regulatory System in Group B Streptococcus Biofilm Formation on Human Fibrinogen.

Streptococcus agalactiae, also known as Group B Streptococcus or GBS, is a commensal colonizer of human vaginal and gastrointestinal tracts that can also be a deadly pathogen for newborns, pregnant women, and the elderly. The SaeRS two-component regulatory system (TCS) positively regulates the expression of two GBS adhesins genes, but its role in the formation of biofilm, an important step in pathogenesis, has not been investigated. In the present study, we set up a novel model of GBS biofilm formation using surfaces coated with human fibrinogen (hFg). Biofilm mass and structure were analyzed by crystal violet staining and three-dimensional fluorescence microscopy, respectively. GBS growth on hFg resulted in the formation of a mature and abundant biofilm composed of bacterial cells and an extracellular matrix containing polysaccharides, proteins, and extracellular DNA (eDNA). Enzymatic and genetic analysis showed that GBS biofilm formation on hFg is dependent on proteins and eDNA in the extracellular matrix and on the presence of covalently linked cell wall proteins on the bacterial surface but not on the type-specific capsular polysaccharide. In the absence of the SaeR regulator of the SaeRS TCS, there was a significant reduction in biomass formation, with reduced numbers of bacterial cells, reduced eDNA content, and disruption of the biofilm architecture. Overall, our data suggest that GBS binding to hFg contributes to biofilm formation and that the SaeRS TCS plays an important role in this process.

Biofilm mass transfer and thermodynamic constraints shape biofilm in trickle bed reactor syngas biomethanation

Syngas (H2, CO2, CO) produced thermochemically from lignocellulosic biomass is an underexploited source for resource recovery and valorisation through its biological conversion for the production of a wide range of chemicals and fuels. Syngas biomethanation is one such promising bioconversion pathway, displaying interesting features such as high conversion efficiency and product selectivity, as methane is the sole product of the process. The biological conversion of syngas to high purity biomethane is still typically associated with a number of challenges related to the syngas composition, CO toxicity, and gas–liquid mass transfer limitations. In this work, the syngas biomethanation process carried out in trickle bed reactors was investigated at its boundary conditions to explore the limits of the process, focusing on syngas composition and mass-transfer conditions potentially limiting process performance. The process was found to be robust when exposed to CO excess, but highly sensitive to H2 excess, which caused severe inhibition even under small amounts of excess H2. This implies that biomethane purity comparable to natural gas can be achieved by addition of renewable H2, but this requires precise control to avoid process failure. Modulating the liquid recirculation rate and gas residence time allowed for a maximum methane productivity of 9.8 ± 0.5 mmol CH4 h−1 Lreactor−1 with full conversion of H2 and CO at a gas residence time of 1 h. Nevertheless, increasing the gas–liquid mass transfer with increasing liquid recirculation rate did not lead to increased methane productivity, which suggested additional rate-limiting bottlenecks in the process. Careful investigation of other factors potentially limiting the process led to the conclusion that diffusive transport of syngas components in the biofilm was the main bottleneck of the process. This diffusive limitation leads to a scenario of severe substrate scarcity in the biofilm phase that conditions the thermodynamic feasibility of the different biochemical reactions involved in syngas biomethanation. In turn, these thermodynamic constraints were found to drive the stratification of microbial groups and sequential consumption of syngas components along the height of the reactor.

Aged Microplastics and Antibiotic Resistance Genes: A Review of Aging Effects on Their Interactions.

Background: Microplastic aging affects the dynamics of antibiotic resistance genes (ARGs) on microplastics, yet no review presents the effects of microplastic aging on the associated ARGs. Objectives: This review, therefore, aims to discuss the effects of different types of microplastic aging, as well as the other pollutants on or around microplastics and the chemicals leached from microplastics, on the associated ARGs. Results: It highlights that microplastic photoaging generally results in higher sorption of antibiotics and ARGs due to increased microplastic surface area and functional group changes. Photoaging produces reactive oxygen species, facilitating ARG transfer by increasing bacterial cell membrane permeability. Reactive oxygen species can interact with biofilms, suggesting combined effects of microplastic aging on ARGs. The effects of mechanical aging were deduced from studies showing larger microplastics anchoring more ARGs due to rough surfaces. Smaller microplastics from aging penetrate deeper and smaller places and transport ARGs to these places. High temperatures are likely to reduce biofilm mass and ARGs, but the variation of ARGs on microplastics subjected to thermal aging remains unknown due to limited studies. Biotic aging results in biofilm formation on microplastics, and biofilms, often with unique microbial structures, invariably enrich ARGs. Higher oxidative stress promotes ARG transfer in the biofilms due to higher cell membrane permeability. Other environmental pollutants, particularly heavy metals, antibacterial, chlorination by-products, and other functional genes, could increase microplastic-associated ARGs, as do microplastic additives like phthalates and bisphenols. Conclusions: This review provides insights into the environmental fate of co-existing microplastics and ARGs under the influences of aging. Further studies could examine the effects of mechanical and thermal MP aging on their interactions with ARGs.

Reactive oxygen species-inducing itraconazole and its anti-biofilm activity against resistant Candida parapsilosis sensu lato biofilm cells isolated from patients with recalcitrant onychomycosis.

Candida parapsilosis was introduced as the second most responsible for nail involvement. The colonization of biotic and abiotic surfaces by Candida spp.can result in the formation of biofilms, which possess a high level of resistance to typical antifungal agents. Since Candida spp. can produce biofilm mass on the surface of the nails, dermatologists should consider appropriate antifungals to eliminate both the planktonic and biofilm cells. The aim of this research was to determine the antifungal efficacy of itraconazole against C. parapsilosis sensu lato biofilm formations, in addition to its static effects. Ten C. parapsilosis sensu lato isolates were enrolled in this study. The use of itraconazole results in the accumulation of reactive oxygen species (ROS) during treatment. In order to verify the correlation between ROS and itraconazole-induced cell death, the viability of cells was analyzed by administering the ROS scavenger Ascorbic acid. The apoptotic features of itraconazole were analyzed using the Annexin V-FITC method. Based on current data, it was found that the generation of intracellular stresses by itraconazole is not observed in cells upon ROS inhibition, emphasizing the importance of intracellular ROS in the apoptotic mechanism of itraconazole. Targeting the oxidative defense system is a powerful point to use ROS-inducing antifungals as a superior choice for more effective therapies in case of recalcitrant onychomycosis.

A novel brominated chalcone derivative as a promising multi-target inhibitor against multidrug-resistant Listeria monocytogenes

Foodborne pathogens continue to challenge public health due to their ability to cause severe illness and their increasing resistance to current antimicrobial treatments. Listeria monocytogenes is a resilient foodborne pathogen that poses significant risks to vulnerable populations, leading to severe infections and high hospitalization rates. The emergence of antimicrobial-resistant (AMR) strains of L. monocytogenes underscores the need for novel therapeutic strategies. In this study, we investigated the antimicrobial efficacy of the (2E)-3-(3,5-dibromo-2-hydroxylphenyl)-1-(5-methylfuran-2-yl) prop-2-en-1-one (DK06) against multidrug-resistant L. monocytogenes. DK06 exhibited a significant dose-dependent inhibition of L. monocytogenes growth, achieving a maximum inhibition of 92.9 % at 320 μM. Molecular docking and dynamics simulations revealed high binding affinities for key virulence proteins PlcB and ArgA, with stable protein-ligand interactions. DK06 also disrupted biofilm formation at sub-MIC levels, reducing extracellular polymeric substances (EPS) and biofilm mass, as observed by scanning electron microscopy (SEM) analysis. Furthermore, DK06 downregulated the expression of virulence genes (plcB, argA, and hly) and decreased hemolytic activity. In vivo zebrafish studies confirmed the safety of DK06 up to 80 μM, demonstrating its efficacy in reducing mortality and oxidative stress associated with L. monocytogenes infection. DK06 also attenuated inflammation by downregulating key inflammatory markers (tnfa, il1b, il6, and nfkb). These findings indicate that DK06 is a promising multi-target inhibitor with potential application in treating infections and combating antimicrobial resistance.

Antibacterial effect and mechanism of theaflavin against Listeria monocytogenes and its application on apple skins.

Theaflavin 3,3'-digallate (TF3), a major polyphenolic component of black tea, exhibits antibacterial effects against many foodborne pathogens. However, the antibacterial mechanisms of TF3 against Listeria monocytogenes remain unclear. In this study, we investigated the effects of TF3 on viability, biofilm, and membrane function of L. monocytogenes by the conventional plating method, crystal violet staining, and microscopy using fluorescent dyes JC-1 and Laurdan, respectively. It was found that TF3 showed excellent antibacterial activity against L. monocytogenes with the minimum inhibitory concentration of 62.5mg/L. The viable count determined on TSA decreased by 3log after the treatment for 2h with TF3 at 62.5mg/L. The viable count determined on TSA containing 4% NaCl decreased by more than 4log after the treatment for 30min with TF3 at the same concentration, suggesting that TF3 gave damage on the cells, enhancing the antibacterial action of 4% NaCl, but the damage was recoverable in the absence of 4% NaCl. To explore the antibacterial mechanisms of TF3, the effects of TF3 on membrane potential and membrane fluidity were investigated. TF3 reduced both membrane potential and fluidity of L. monocytogenes at 62.5mg/L, suggesting that TF3 damaged the structural integrity of the cell membrane. TF3 reduced biofilm mass of mature biofilm of L. monocytogenes. Moreover, THEAFLAVIN TF40, a commercially available Camellia sinensis leaf extract containing TF3, reduced viable count of L. monocytogenes by 2log on apple skin. These results suggest the potential of theaflavins as a natural anti-Listeria disinfectant.

Antibacterial and Anti-Biofilm Effect of Silver Nanoparticles Synthesized from Origanum majorana and Echinacea purpurea (L.) Moench Plants via Green Synthesis

Objective: Nanotechnology has gained importance in the fight against epidemics and antibiotic resistance. Nanotechnology is a potential way to prevent the increase of multidrug-resistant bacterial species. The aim of this study was to determine the antimicrobial and anti-biofilm activities of Origanum majorana and Echinacea purpurea silver nanoparticles (AgNPs) against Staphylococcus aureus (S.aureus) ATCC 25923, Escherichia coli (E.coli) 25922, Pseudomonas aureginosa (P.aureginosa) 27853, Klebsiella pneumoniae (K. pneumoniae) 700603. Methods: Minimum Inhibitory Concentration (MIC) values of silver nanoparticles were determined by microdilution method in 96-well ELISA plates. The anti-biofilm effect of silver nanoparticles was performed by crystal violet method in 96-well microplates. Results: The MIC value of four isolates was determined as 128 µg/ml for two nanoparticles. Only 256 µg/ml and 512 µg/ml were found for S. aureus. In vitro, anti-biofilm effect of AgNPs against biofilm forming bacteria was evaluated in a dose-dependent manner. S. aureus, AgNP synthesized from Origanum majorana plant extract (512 µg/ml) reduced biofilm formation by 92% after 24 hours of incubation. As a result of 24 hours incubation of S. aureus with AgNP (512 µg/ml) synthesized from Echinacea purpurea (L.) It was determined that biofilm formation decreased by 85%. It was observed that both different nanoparticles significantly inhibited the biofilm mass. Conclusion: AgNPs showed antimicrobial and antibiofilm effects for standard strains. The use of AgNPs as antimicrobials is promising for the future.

Antibiofilm Effect of Sequential Application of Ozonated Water, Acetic Acid and Lactic Acid on Salmonella Typhimurium and Staphylococcus aureus Biofilms In Vitro

Biofilms are highly resistant to disinfectants and antimicrobials and are known as the primary source of food contamination. Salmonella Typhimurium (S. Typhimurium) and Staphylococcus aureus (S. aureus) have an excellent ability to form biofilm. This study aimed to evaluate the antibiofilm activity of ozonated water (O), acetic acid (AA), and lactic acid (LA), individually and sequentially, against biofilms of S. Typhimurium and S. aureus formed on the polystyrene surfaces. The antibiofilm effects of the treatments were evaluated using crystal violet staining and the viable count determination methods. In the staining method, the highest percentage of biofilm mass reduction was induced by successive use of ozonated water and acetic acid (O-AA), which reduced S. aureus biofilm mass by 44.36%. The sequential use of ozonated water and lactic acid (O-LA) could decrease S. Typhimurium biofilm mass by 57.26%. According to the viable count method, the most effective treatment was the sequential use of ozonated water and lactic acid (O-LA), which reduced S. aureus and S. Typhimurium biofilms by 1.76 and 4.06 log, respectively. It was concluded that the sequential use of ozonated water and organic acids can be considered a practical and environmentally friendly approach to control biofilms.

Totarol exhibits antibacterial effects through antibiofilm and combined interaction against vancomycin-resistant Enterococcus faecalis.

The rise of vancomycin-resistant enterococci (VRE) due to antibiotic overuse poses a significant threat to long-term care patients and those with impaired immune systems. Therefore, it is imperative to seek alternatives to overcome multidrug resistance. This study aimed to evaluate totarol, a natural compound derived from Podocarpus totara, for its antibacterial activity against vancomycin-resistant Enterococcus faecalis (VREF). Totarol exhibited potent antibacterial activity at a very low concentration of 0.25µg/mL and demonstrated antibiofilm effects through biofilm inhibitory concentration and biofilm eradication concentration assays. Confocal laser scanning microscopy confirmed that totarol inhibited not only biofilm mass but also bacterial cell viability. The combinatorial use of sublethal concentrations of totarol and vancomycin showed antibacterial activity, as observed in the time-kill assay. Quantitative polymerase chain reaction assays revealed a concentration-dependent downregulation of key virulence genes (vanA, ace, asa, efaA, and esp) in VREF when exposed to totarol. In summary, totarol emerges as a promising adjuvant with vancomycin for inhibiting VREF, addressing vancomycin resistance and biofilm formation-critical challenges associated with VRE infection. Since this was an in vitro study, the role of totarol in the clinical implications of VREF treatment remains to be demonstrated.

Pinhão potential and their parts (failures, shells, and almonds) in the elaboration of yogurts containing acai pulp: physicochemical, nutritional, and functional properties, antimicrobial activity, and multi-elemental profile

This study applies natural resources, prioritizing recyclable and renewable inputs produced by pinhão cultivation, whose purpose is to use the failures, shells, and almonds as a source of bioactive compounds addition in yogurt, ensuring intelligent use of these natural resources. Thus, one açaí yogurt sample and eight yogurt formulations containing portions of pinhão byproducts between 5 % and 10 % were elaborated. These formulations were compared regarding their physicochemical, nutritional, functional properties, antimicrobial activity, and multi-elemental profile properties. Enriching açaí yogurt with pinhão byproducts does not significantly differ in protein, lipid, moisture, and mineral salt content between all samples with pinhão byproducts. Açaí yogurts enriched with pinhão byproducts had 5.71 to 26.07 % times total protein than the control sample, and total fiber also had a significant increase in samples ranging between 18.62 to 85.29 % times more than the control sample. Regarding color settings, all yogurt samples tended to be red–purple. A sample of açaí yogurt with pine nut flour and whole pine nut flour caused a biofilm mass amount of 46.58, 45.55, and 11.85 % for Listeria monocytogenes, Salmonella enteritidis and Pseudomonas aeruginosa. The behavior of pathogenic bacteria is related to the total polyphenol content in yogurts enriched with pinhão byproducts, which increased from 8.27 to 18.24 mg/100 g. Yogurt with açaí enriched with whole pinhão flour showed high antioxidant capacity. The sample’s antioxidant activity results increased by 47.62 % and 130.38 % in the ABTS and DPPH analyses, respectively. The compounds in pinhão failure nanosuspensions, pinhão flour, whole pinhão flour, and yogurts were identified and divided into hydrophilic and lipophilic classes. Five classes (amino acids, organic acids, sugars, phenols, and cyclitols) were identified as hydrophilic. Lipophilic compounds were identified and separated into six classes (carboxylic acids, diterpenes, alcohols, Α-hydroxy acids, sterols, and triterpenes). The addition of pinhão byproducts increased the contents of Ca, Fe, K, Na, and P. Açaí yogurt with pinhão nanosuspension, pinhão flour, and whole pinhão flour had the highest Ca content (2164.38 ± 2.16 µg/L). Açaí yogurt with pinhão flour and whole pinhão flour had the highest Fe content (84.02 ± 0.08 µg/L).

Oxygen Microenvironments in E. coli Biofilm Nutrient Transport Channels: Insights from Complementary Sensing Approaches.

Chemical gradients and the emergence of distinct microenvironments in biofilms are vital to the stratification, maturation and overall function of microbial communities. These gradients have been well characterised throughout the biofilm mass but the microenvironment of recently discovered nutrient transporting channels in Escherichia coli biofilms remains unexplored. This study employs three different oxygen sensing approaches to provide a robust quantitative overview of the oxygen gradients and microenvironments throughout the biofilm transport channel networks formed by E. coli macrocolony biofilms. Oxygen nanosensing combined with confocal laser scanning microscopy established that the oxygen concentration changes along the length of biofilm transport channels. Electrochemical sensing provided precise quantification of the oxygen profile in the transport channels, showing similar anoxic profiles compared with the adjacent cells. Anoxic biosensing corroborated these approaches, providing an overview of the oxygen utilisation throughout the biomass. The discovery that transport channels maintain oxygen gradients contradicts the previous literature that channels are completely open to the environment along the apical surface of the biofilm. We provide a potential mechanism for the sustenance of channel microenvironments via orthogonal visualisations of biofilm thin sections showing thin layers of actively growing cells. This complete overview of the oxygen environment in biofilm transport channels primes future studies aiming to exploit these emergent structures for new bioremediation approaches.

Biofilm inhibition of denture cleaning tablets and carvacrol on denture bases produced with different techniques

ObjectivesThis study compares the biofilm inhibition effects of denture cleaning tablets, carvacrol, and their combined use against Candida albicans on denture bases produced with different techniques. Additionally, the surface roughness and contact angles of these denture bases were evaluated.Materials and methodsTest samples were prepared from four different denture base materials (cold-polymerized, heat-polymerized, CAD/CAM milling, and 3D-printed). The surface roughness and contact angles of the test samples were measured using a profilometer and goniometer, respectively. For the evaluation of biofilm inhibition, samples were divided into 5 subgroups: Corega and carvacrol, separately and combined treatments, positive (inoculated with C. albicans) and negative control (non-inoculated with C. albicans, only medium). Biofilm mass was determined using the crystal violet method. An additional prepared test sample for each subgroup was examined under scanning electron microscopy (SEM).ResultsThe surface roughness values of the 3D-printed test samples were found to be statistically higher than the other groups (P < .001). The water contact angle of all test materials was not statistically different from each other (P > .001). Corega and carvacrol, separately and combined, significantly decreased the amount of biofilm on all surfaces (P < .0001). Treatment of corega alone and in combination with carvacrol to the 3D-printed material caused less C. albicans inhibition than the other groups (P < .001; P < .05).ConclusionsThe surface roughness values of all test groups were within the clinically acceptable threshold. Although Corega and carvacrol inhibited C. albicans biofilms, their combined use did not show a synergistic effect.Clinical relevanceCarvacrol may be used as one of the disinfectant agents for denture cleaning due to its biofilm inhibition property.

FTY720 Reduces the Biomass of Biofilms in Pseudomonas aeruginosa in a Dose-Dependent Manner.

Pseudomonas aeruginosa, a nosocomial pathogen, has strong biofilm capabilities, representing the main source of infection in the human body. Repurposing existing drugs has been explored as an alternative strategy to combat emerging antibiotic-resistant pathogens. Fingolimod hydrochloride (FTY720), an immunomodulatory drug for multiple sclerosis, has shown promising antimicrobial effects against some ESKAPE pathogens. Therefore, the effects of FTY720 on the biofilm capabilities of Pseudomonas aeruginosa were investigated in this study. It was determined that FTY720 inhibited the growth of P. aeruginosa PAO1 at 100 µM. The significant reduction in PAO1 cell viability was observed to be dose-dependent. Additional cytotoxicity analysis on human cell lines showed that FTY720 significantly reduced viabilities at sub-inhibitory concentrations of 25-50 µM. Microtiter assays and confocal analysis confirmed reductions in biofilm mass and thickness and the cell survivability ratio in the presence of FTY720. Similarly, virulence production and biofilm-related gene expression (rhlA, rhlB, pilA, pilI, fliC, fliD and algR) were determined. The results demonstrate that pigment production was affected and quantitative real-time PCR analysis showed a variable degree of reduced gene expression in response to FTY720 at 12.5-50 µM. These findings suggest that FTY720 could be repurposed as an alternative antibiofilm agent against Pseudomonas aeruginosa.

Response and recovery of nitrifying moving bed biofilm reactor systems exposed to 1°C with varying levels of ammonia starvation

This study investigated the impact of varying total ammonia nitrogen (TAN) feed levels along with water temperature decreases on the performance of nitrifying moving bed biofilm reactor (MBBR) at 1 °C and its recovery at 3 °C. Five MBBR reactors were operated with different TAN concentrations as water temperature decreased from 20 to 3 °C: reactor R1 at 30 mg N/L, reactor R2 at 20 mg N/L, reactor R3 at 15 mg N/L, reactor R4 at 10 mg N/L and reactor R5 at 0 mg N/L. The corresponding biofilm characteristics were also analyzed to understand further nitrifying MBBR under different TAN feeding scenarios. The findings revealed that the higher TAN levels were before reaching 1 °C, the better nitrification performance and the more biomass grew. However, the highest TAN concentration (30 mg N/L) might negatively affect the nitrification performance, the activity of nitrifiers, and the growth of biofilms at 1 °C because of the toxic effects of un-ionized or free ammonia (FA). It was observed that the activities of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were affected by FA concentrations ranging from 0.2 to 0.7 mg N/L at 1 °C, but they could gradually be adapted to such inhibitory environment, with NOB recovering more quickly and robustly than AOB. The study identified 20 mg N/L (67 % of maximum influent TAN at 1 °C in R2 as the optimal TAN feeding concentration, achieving over 90 % TAN removal and a surface area removal rate (SARR) of 0.78 ± 0.02 g N/m2·d at 1 °C. Meanwhile, R2 also exhibited the highest biofilm mass, with total solids at 13.3 mg/carrier and volatile solids at 11.3 mg/carrier. As TAN was removed, nitrite accumulation was observed at 1 °C, and higher influent TAN concentrations prior to 1 °C appeared to delay the accumulation. When water temperature increased from 1 °C to 3 °C, nitrification performance improved significantly in all reactors without nitrite accumulation, and the higher TAN feeding in the previous stage led to faster recovery. Compared with 20 °C, biofilm became thinner and denser at 1 °C and 3 °C. Furthermore, this study revealed significant shifts in microbial community composition and nitrifier abundances in response to changes in water temperature and influent TAN levels. The dominant nitrifiers were identified as Nitrosomonadaceae (AOB) and Nitrospiraceae (NOB). At 1 °C, the nitrifier abundances were significantly correlated with SARRs, FA, and biofilm density. R2, which exhibited the best nitrification performance, maintained higher nitrifier abundances at 1 °C.

Characterization of structure of peptidyl-tRNA hydrolase from Enterococcus faecium and its inhibition by a pyrrolinone compound

In bacteria, peptidyl-tRNA hydrolase (Pth, E.C. 3.1.1.29) is a ubiquitous and essential enzyme for preventing the accumulation of peptidyl-tRNA and sequestration of tRNA. Pth is an esterase that cleaves the ester bond between peptide and tRNA. Here, we present the crystal structure of Pth from Enterococcus faecium (EfPth) at a resolution of 1.92 Å. The two molecules in the asymmetric unit differ in the orientation of sidechain of N66, a conserved residue of the catalytic site. Enzymatic hydrolysis of substrate α-N-BODIPY-lysyl-tRNALys (BLT) by EfPth was characterized by Michaelis-Menten parameters KM 163.5 nM and Vmax 1.9 nM/s. Compounds having pyrrolinone scaffold were tested for inhibition of Pth and one compound, 1040-C, was found to have IC50 of 180 nM. Antimicrobial activity profiling was done for 1040-C. It exhibited equipotent activity against drug-susceptible and resistant S. aureus (MRSA and VRSA) and Enterococcus (VSE and VRE) with MICs 2–8 μg/mL. 1040-C synergized with gentamicin and the combination was effective against the gentamicin resistant S. aureus strain NRS-119. 1040-C was found to reduce biofilm mass of S. aureus to an extent similar to Vancomycin. In a murine model of infection, 1040-C was able to reduce bacterial load to an extent comparable to Vancomycin.