Inactivation of Pseudomonas aeruginosa biofilms formed under high shear stress on various hydrophilic and hydrophobic surfaces by a continuous flow of ozonated water

Abstract

The inactivation of Pseudomonas aeruginosa biofilms grown on glass under high shear stress and exposed to a range of dissolved ozone concentrations (2, 5 and 7?ppm) at 10 and 20?min was investigated. The regression equation, log reduction (biofilm)?=?0.64?+?0.59×(C – 2)?+?0.33×(T – 10), described the dependence of biofilm inactivation on the dissolved ozone concentration (C, ppm) and contact time (T, min). The predicted D-values were 11.1, 5.7 and 2.2?min at 2, 5 and 7?ppm, respectively. Inactivation of biofilms grown on various surfaces was tested at a single dissolved ozone concentration of 5?ppm and a single exposure time of 20?min. Biofilms grown on plastic materials showed inactivation results similar to that of biofilms on glass, while biofilms grown on ceramics were statistically significantly more difficult to inactivate, suggesting the importance of utilizing non-porous materials in industrial and clinical settings.     

Gas-phase removal of biofilms from various surfaces using carbon dioxide aerosols

The present study evaluated the removal of Escherichia coli XL1-blue biofilms using periodic jets of carbon dioxide aerosols (a mixture of solid and gaseous CO(2)) with nitrogen gas. The aerosols were generated by the adiabatic expansion of high-pressure CO(2) gas through a nozzle and used to remove air-dried biofilms. The areas of the biofilms were measured from scanning electron micrographs before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured with various air-drying times of the biofilms before the treatment, surface materials, and durations of CO(2) aerosols in each 8-s aerosol-nitrogen cleaning cycle. Nearly 100% of the fresh biofilms were removed from the various surfaces very reliably within 90?s. This technique can be useful for removing unsaturated biofilms on solid surfaces and has potential applications for cleaning bio-contaminated surfaces.     

Removal of Burkholderia cepacia biofilms with oxidants

Iodine is used to disinfect the water system aboard US space shuttles and is the anticipated biocide for the international space station. Water quality on spacecraft must be maintained at the highest possible levels for the safety of the crew. Furthermore, the treatment process used to maintain the quality of water on research must be robust and operate for long periods with minimal crew intervention. Biofilms are recalcitrant and pose a major threat with regard to chronic contamination of spacecraft water systems. We measured the effectiveness of oxidizing biocides on the removal and regrowth of Burkholderia (Pseudomonas) cepacia biofilms. B. cepacia, isolated from the water distribution system of the space shuttle Discovery, was grown in continuous culture to produce a bacterial contamination source for biofilm formation and removal studies. A 10(7) CFU ml-1 B. cepacia suspension, in distilled water, was used to form biofilms on 3000 micrometers2 glass surfaces. Rates of attachment were measured directly with image analysis and were found to be 7.8, 15.2, and 22.8 attachment events h-1 for flow rates of 20.7, 15.2, and 9.8 ml min-1, respectively. After 18 h of formation, the B. cepacia biofilms were challenged with oxidants (ozone, chlorine, and iodine) and the rates of biofilm removal determined by image analysis. Fifty percent of the biofilm material was removed in the first hour of continous treatment with 24 mg l-1 chlorine or 2 mg l-1 ozone. Iodine (48 mg l-1) did not remove any measurable cellular material after 6 h continuous contact. After this first removal of biofilms by the oxidants, the surface was allowed to refoul and was again treated with the biocide. Iodine was the only compound that was unable to remove cellular debris from either primary or secondary biofilms. Moreover, treating primary biofilms with iodine increased the rate of formation of secondary biofilms, from 4.4 to 5.8 attachment events h-1. All the oxidants tested inactivated the B. cepacia associated with both primary and secondary biofilms. The amount of biocide needed to inactivate 50% of planktonic B. cepacia in 10 min at 25 degrees C was 8.4, 0.5, and 0.2 mg l-1 for iodine, chlorine, and ozone, respectively. The data suggest that iodine maynot be the best chemical for treating of biofilms when removal of cellular material is required.     

Effect of intravenous vitamin C on cytokine activation and oxidative stress in end-stage renal disease patients receiving intravenous iron sucrose

Reticuloendothelial blockade in hemodialysis patients prevents optimal intravenous (IV) iron utilization. Vitamin C has emerged as a potential therapy to improve anemia treatment by enhancing iron mobilization. However, Vitamin C can act as a pro-oxidant in the presence of iron. This was a prospective, open-label, crossover study. Thirteen patients with end-stage renal disease on hemodialysis and four healthy controls were assigned to receive 100?mg of IV iron sucrose (IS) or 100?mg of IV IS co-administered with 300?mg of IV Vitamin C (IS?+?C) in random sequence. Serum samples for IL-1, IL-6, TNF-α and IL-10 and non-transferrin bound iron were obtained at baseline, 45?min and 105?min post study medication administration. Peripheral blood mononuclear cells were isolated at the same time points and stained with fluorescent probes to identify intracellular reactive oxygen species and mitochondrial membrane potential (Δψm) by flow cytometry. Lipid peroxidation was assessed by plasma F2-isoprosatane concentration. Both IS and IS?+?C were associated with increased plasma F2-isoprostanes concentrations post-infusion. Maximal plasma F2-isoprostane concentrations after IS?+?C were significantly elevated from baseline (234?±?0.04 vs. 0.198?±?0.028?ng/mL, p?=?0.02). After IS?+?C, IL-1, IL-6, IL-10, and TNF-alpha were significantly elevated compared to baseline. After IS alone only IL-6 was noted to be elevated. Intracellular production of H(2)O(2) and loss of mitochondrial membrane potential (Δψm) was observed after IS while IS?+?C was associated with increased O (2) (·-) production. Both IS and IS?+?C induced serum cytokine activation accompanied by lipid peroxidation, however, IS?+?C induced higher plasma concentrations of F2-isoprostanes, IL-1, IL-10, and TNF-α post-infusion. Long-term safety studies of IV iron co-administered with Vitamin C are warranted.     

Effect of chlorination on the development of marine biofilms dominated by diatoms

This study addressed the antifouling efficiency of commercially available chlorine at different concentrations (0.5%, 1%, and 2%) and exposure times (0.5?min, 1?min, 5?min, and 15 min). The rapid and non-destructive FIRe (fluorescence induction and relaxation) technique was used to evaluate the effects of the biocide on diatom dominated biofilms. The efficiency of chlorine in removing diatoms from the developed biofilms increased with an increase in concentration and exposure time. The fluorescence measurements revealed low F(v)/F(m) and high σ(PSII) values for chlorine-treated Navicula and Amphora biofilms indicating that chlorination was efficient in damaging the photosystem-II reaction centers. Chlorination also caused mortality of diatom cells by damaging the cell body. In natural biofilms, the biocidal effect of chlorine was species specific; species of Amphiphrora, Navicula, Cylindrotheca, and Coscinodiscus showed an increase in the density of the population, but species of Pleurosigma, Amphora, and Thalassionema did not increase in density after chlorine treatment. It was also demonstrated that diatoms can colonize, grow and photosynthesize on chlorine-treated surfaces. Under pulse chlorination (treatment every 6 h), irrespective of chlorine concentration, the development of biofouling decreased with an increase in exposure time. Differences between exposure times of 1 to 15?min were not significant. Additionally, transmission levels of the control (non-chlorine-treated) fouled coupons reduced significantly (～20%) compared to the chlorine-treated fouled coupons (<2%). These results suggest that chlorine can be used as a biocide to control the development of diatom biofilms.     

Removal of different-age biofilms using carbon dioxide aerosols

Many techniques to inactivate or remove biofilms in a wide variety of applications have been developed. Most of these techniques have been applied to biofilms at their initial stage of growth, since they are generally difficult to eradicate once established. The removal of established biofilms has received relatively little attention. In this paper, we report the effectiveness of periodic jets of carbon dioxide aerosols (a mixture of solid and gaseous CO₂) to remove Escherichia coli (XL1-blue) biofilms of different ages (up to 3 weeks) on silicon surfaces. The biofilms were not immersed in liquids after growth/rinsing and were treated with the CO₂ aerosols. The CO₂ aerosols were generated by the adiabatic expansion of high-pressure CO2 gas through a nozzle. The surface area of the biofilms was measured from fluorescent images before and after applying the aerosols for 11, 20, and 30 cycles (each cycle: 8 sec), to compute the removal efficiency. The removal efficiencies decreased with increasing growth time and for the 3-week-old biofilms, they ranged from 91.5 to 99.6% within 4 min. This technique was highly effective for removing both fresh and old biofilms, but some of the biofilm debris such as growth media remained. Further, this CO₂ aerosol technique was compared with other removal techniques.     

A sustainable process for adsorptive removal of methylene blue onto a food grade mucilage: kinetics,thermodynamics, and equilibrium evaluation

Abstract

Adsorption of dyes onto natural materials like polysaccharides is considered a green chemistry approach for remediation of wastewater. In this work, the polysaccharide isolated from the corm of Colocasia esculenta (L.) Schott or taro tuber (CEM) was utilized for removing methylene blue (MB) from aqueous solution by batch adsorption method. The CEM adsorbent was characterized by FTIR spectroscopy, Brunauer–Emmett–Teller (BET), and scanning electron microscopy (SEM). The solution pH and adsorbent dose have been found to have a significant positive correlation with the adsorptive removal efficiency of CEM for MB dye. The removal efficiency of CEM was found to be 72.35% under the optimum conditions; 20?mg/L initial concentration of dye, 120?mg of adsorbent dose, solution pH 8.5, 311.2?K temperature and 80?min contact time. The adsorption of MB onto CEM followed best the Freundlich isotherm and pseudo-second-order kinetics. The adsorption was thermodynamically favorable and was endothermic in nature. The desorption/adsorption data justifiably indicated the reuse capability of CEM adsorbent for MB adsorption. Hence, CEM may be regarded as an eco-friendly and cost-effective natural adsorbent for MB dye removal from aqueous solution.     

The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l(-1) caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.     

A synergistic d-tyrosine and tetrakis hydroxymethyl phosphonium sulfate biocide combination for the mitigation of an SRB biofilm

Microbiologically influenced corrosion (MIC) is a major problem in various industries such as oil and gas, and water utilities. Billions of dollars are lost to microbiologically influenced corrosion (MIC) each year in the US. The key to MIC control is biofilm mitigation. Sulfate-reducing bacteria (SRB) are often the culprits. They are also involved in souring and biofouling. SRB biofilms are notoriously difficult to eradicate. Due to environmental concerns and increasing costs, better biocide treatment strategies are desired. Recent studies suggested that D: -tyrosine and some other D: -amino acids may signal biofilm dispersal. Experimental results in this work indicated that D: -tyrosine is an effective biocide enhancer for tetrakis hydroxymethyl phosphonium sulfate (THPS) that is a green biocide. Desulfovibrio vulgaris (ATCC 7757) was used in biofilm prevention and biofilm removal tests. It was found that 100?ppm D: -tyrosine alone and 50?ppm THPS alone were both ineffective against the SRB biofilm. However, when 1?ppm D: -tyrosine was combined with 50?ppm THPS, the synergy between the two chemicals successfully prevented the establishment of the SRB biofilm on C1018 mild steel coupon surfaces in batch treatment tests. It also eradicated established SRB biofilms from coupon surfaces in both 1 and 3-h shock treatment tests.     

Hydrodynamic deformation and removal of Staphylococcus epidermidis biofilms treated with urea, chlorhexidine, iron chloride, or DispersinB

The force-deflection and removal characteristics of bacterial biofilm were measured by two different techniques before and after chemical, or enzymatic, treatment. The first technique involved time lapse imaging of a biofilm grown in a capillary flow cell and subjected to a brief shear stress challenge imparted through increased fluid flow. Biofilm removal was determined by calculating the reduction in biofilm area from quantitative analysis of transmission images. The second technique was based on micro-indentation using an atomic force microscope. In both cases, biofilms formed by Staphylococcus epidermidis were exposed to buffer (untreated control), urea, chlorhexidine, iron chloride, or DispersinB. In control experiments, the biofilm exhibited force-deflection responses that were similar before and after the same treatment. The biofilm structure was stable during the post-treatment shear challenge (1% loss). Biofilms treated with chlorhexidine became less deformable after treatment and no increase in biomass removal was seen during the post-treatment shear challenge (2% loss). In contrast, biofilms treated with urea or DispersinB became more deformable and exhibited significant biofilm loss during the post-treatment flow challenge (71% and 40%, respectively). During the treatment soak phase, biofilms exposed to urea swelled. Biofilms exposed to iron chloride showed little difference from the control other than slight contraction during the treatment soak. These observations suggest the following interpretations: (1) chemical or enzymatic treatments, including those that are not frankly antimicrobial, can alter the cohesion of bacterial biofilm; (2) biocidal treatments (e.g., chlorhexidine) do not necessarily weaken the biofilm; and (3) biofilm removal following treatment with agents that make the biofilm more deformable (e.g., urea, DispersinB) depend on interaction between the moving fluid and the biofilm structure. Measurements such as those reported here open the door to development of new technologies for controlling detrimental biofilms by targeting biofilm cohesion rather than killing microorganisms.     

Photomechanical wave-assisted molecular delivery in oral biofilms

Photomechanical waves (PW), the product of an intense light beam interaction with a target material, enhance molecular delivery across biological membranes and skin. The ability to deliver methylene blue (MB), a fluorescent probe and photosensitizer, into bacterial biofilms was demonstrated by applying PW on saliva-derived multi-species biofilms that were developed on agar surfaces in 24-well plates. PW were generated with a Q-switched Nd:YAG laser and were directed into the biofilms in the presence of 25 μg/ml MB. The biofilms were then irradiated with red light at 665 nm. After illumination, adherent bacteria were scraped and spread over the surface of blood agar plates. Survival fractions were calculated by counting bacterial colonies. Microbial analysis was performed via a colony lift method and a DNA checkerboard assay using whole genomic probes to 40 oral microorganisms. Visual analysis by confocal scanning laser microscopy demonstrated that the application of PW enhanced the penetration depth of MB in biofilms. Exposure to MB, PW and light led to a significant reduction of the mean levels of log₁₀ CFU counts compared with the group that received MB and light (P = 0.006). The DNA checkerboard assay showed some benefit from PW-assisted phototargeting in 25 biofilm microorganisms relative to phototreatment alone. Our data provide a basis for further exploration and optimization of PW parameters for complete eradication of microorganisms in oral microcosm biofilms.     

The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l^?1 caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.     

Gas-phase removal of biofilms from various surfaces using carbon dioxide aerosols

The present study evaluated the removal of Escherichia coli XL1-blue biofilms using periodic jets of carbon dioxide aerosols (a mixture of solid and gaseous CO₂) with nitrogen gas. The aerosols were generated by the adiabatic expansion of high-pressure CO₂ gas through a nozzle and used to remove air-dried biofilms. The areas of the biofilms were measured from scanning electron micrographs before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured with various air-drying times of the biofilms before the treatment, surface materials, and durations of CO₂ aerosols in each 8-s aerosol–nitrogen cleaning cycle. Nearly 100% of the fresh biofilms were removed from the various surfaces very reliably within 90 s. This technique can be useful for removing unsaturated biofilms on solid surfaces and has potential applications for cleaning bio-contaminated surfaces.     

Antimicrobial activity and effectiveness of a combination of sodium hypochlorite and hydrogen peroxide in killing and removing Pseudomonas aeruginosa biofilms from surfaces

AIMS: To evaluate both the antimicrobial activity and the effectiveness of a combination of sodium hypochlorite and hydrogen peroxide (Ox-B) for killing Pseudomonas aeruginosa ATCC 19142 cells and removing P. aeruginosa biofilms on aluminum or stainless steel surfaces. METHODS AND RESULTS: Pseudomonas aeruginosa biofilms were developed in tryptic soy broth containing vertically suspended aluminium or stainless steel plates. Biofilms were exposed to a mixed sodium hypochlorite and hydrogen peroxide solution as a sanitizer for 1, 5 and 20 min. The sanitizer was then neutralized, the cells dislodged from the test surfaces, and viable cells enumerated. Cell morphologies were determined using scanning (SEM) and transmission electron microscopy (TEM). Cell viability was determined by confocal scanning laser microscopy (CSLM). Biofilm removal was monitored by Fourier transform infrared (FTIR) spectrophotometry. Cell numbers were reduced by 5-log to 6-log after 1 min exposure and by 7-log after 5 min exposure to Ox-B. No viable cells were detected after a 20 min exposure. Treatment with equivalent concentrations of sodium hypochlorite reduced viable numbers by 3-log to 4-log after 1 min exposure and by 4-log to 6-log after 5 min, respectively. A 20 min exposure achieved a 7-log reduction. Hydrogen peroxide at test concentration treatments showed no effect. FTIR analysis of treated pseudomonad biofilms on aluminium or stainless steel plates showed either a significant reduction or complete removal of biofilm material after a 5 min exposure to the mixed sodium hypochlorite and hydrogen peroxide solution. SEM and TEM images revealed damage to cell wall and cell membranes. CONCLUSIONS: A combination of sodium hypochlorite and hydrogen peroxide effectively killed P. aeruginosa cells and removed biofilms from both stainless steel and aluminium surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of sodium hypochlorite and hydrogen peroxide can be used as an alternative disinfectant and/or biofilm remover of contaminated food processing equipment.     

Rapid methods to assess sanitizing efficacy of benzalkonium chloride to Listeria monocytogenes biofilms

Different methods were used to investigate biofilm growth including crystal violet staining, ATP bioluminescence and total viable count. Seven strains of Listeria monocytogenes and 8 of their derivative strains were screened for their capacity to form biofilms. Both adaptation to benzalkonium chloride (BC) and curing of plasmids did not significantly affect biofilm-forming ability. The strains of L. monocytogenes belonging to serotype 1 formed biofilms significantly better as compared to serotype 4 (P=0.0003). To estimate the efficacy of BC for biofilm elimination the best and the poorest biofilm-formers were used (C719 and LJH 381). It was observed that, L. monocytogenes strain C719 in biofilms is at least 1000 times more resistant to BC than in planktonic form. Cells present in biofilms were shown to recover and grow after BC treatment thus providing a source of recontamination. It was shown that ATP bioluminescence provides good correlation with bacterial counts of L. monocytogenes in biofilms. Staining with crystal violet, on the contrary, did not correlate with bacterial growth in biofilms in the presence of high concentrations of BC but provided information on the concentration of bacterial cells, both live and dead, attached to the surface. ATP bioluminescence was found to be a reliable method for rapid estimation of the efficacy of sanitizers for biofilm disinfection. Crystal violet staining, on the other hand, was shown to be a suitable method to monitor removal of biofilms. Our investigation showed that for Listeria biofilms concentrations of BC higher then 10 mg/ml should be applied for at least 30 min to kill almost all the live cells in biofilms. However, this concentration was still not enough to remove biofilms from the surface of plastic.     

A Biofilm Pocket Model to Evaluate Different Non-Surgical Periodontal Treatment Modalities in Terms of Biofilm Removal and Reformation,Surface Alterations and Attachment of Periodontal Ligament Fibroblasts

Background and Aim

There is a lack of suitable in vitro models to evaluate various treatment modalities intending to remove subgingival bacterial biofilm. Consequently, the aims of this in vitro-study were: a) to establish a pocket model enabling mechanical removal of biofilm and b) to evaluate repeated non-surgical periodontal treatment with respect to biofilm removal and reformation, surface alterations, tooth hard-substance-loss, and attachment of periodontal ligament (PDL) fibroblasts.

Material and Methods

Standardized human dentin specimens were colonized by multi-species biofilms for 3.5 days and subsequently placed into artificially created pockets. Non-surgical periodontal treatment was performed as follows: a) hand-instrumentation with curettes (CUR), b) ultrasonication (US), c) subgingival air-polishing using erythritol (EAP) and d) subgingival air-polishing using erythritol combined with chlorhexidine digluconate (EAP-CHX). The reduction and recolonization of bacterial counts, surface roughness (Ra and Rz), the caused tooth substance-loss (thickness) as well as the attachment of PDL fibroblasts were evaluated and statistically analyzed by means of ANOVA with Post-Hoc LSD.

Results

After 5 treatments, bacterial reduction in biofilms was highest when applying EAP-CHX (4 log10). The lowest reduction was found after CUR (2 log10). Additionally, substance-loss was the highest when using CUR (128±40 µm) in comparison with US (14±12 µm), EAP (6±7 µm) and EAP-CHX (11±10) µm). Surface was roughened when using CUR and US. Surfaces exposed to US and to EAP attracted the highest numbers of PDL fibroblasts.

Conclusion

The established biofilm model simulating a periodontal pocket combined with interchangeable placements of test specimens with multi-species biofilms enables the evaluation of different non-surgical treatment modalities on biofilm removal and surface alterations. Compared to hand instrumentation the application of ultrasonication and of air-polishing with erythritol prevents from substance-loss and results in a smooth surface with nearly no residual biofilm that promotes the reattachment of PDL fibroblasts.     

Pseudomonas aeruginosa Biofilm Response and Resistance to Cold Atmospheric Pressure Plasma Is Linked to the Redox-Active Molecule Phenazine

Pseudomonas aeruginosa is an important opportunistic pathogen displaying high antibiotic resistance. Its resistance is in part due to its outstanding ability to form biofilms on a range of biotic and abiotic surfaces leading to difficult-to-treat, often long-term infections. Cold atmospheric plasma (CAP) is a new, promising antibacterial treatment to combat antibiotic-resistant bacteria. Plasma is ionized gas that has antibacterial properties through the generation of a mix of reactive oxygen and nitrogen species (RONS), excited molecules, charged particles and UV photons. Our results show the efficient removal of P. aeruginosa biofilms using a plasma jet (kINPen med), with no viable cells detected after 5 min treatment and no attached biofilm cells visible with confocal microscopy after 10 min plasma treatment. Because of its multi-factorial action, it is widely presumed that the development of bacterial resistance to plasma is unlikely. However, our results indicate that a short plasma treatment (3 min) may lead to the emergence of a small number of surviving cells exhibiting enhanced resistance to subsequent plasma exposure. Interestingly, these cells also exhibited a higher degree of resistance to hydrogen peroxide. Whole genome comparison between surviving cells and control cells revealed 10 distinct polymorphic regions, including four belonging to the redox active, antibiotic pigment phenazine. Subsequently, the interaction between phenazine production and CAP resistance was demonstrated in biofilms of transposon mutants disrupted in different phenazine pathway genes which exhibited significantly altered sensitivity to CAP.     

Destruction of oral biofilms formed in situ on machined titanium (Ti) surfaces by cold atmospheric plasma

The decontamination of implant surfaces represents the basic procedure in the management of peri-implant diseases, but it is still a challenge. The study aimed to evaluate the degradation of oral biofilms grown in situ on machined titanium (Ti) discs by cold atmospheric plasma (CAP). ～ 200 Ti discs were exposed to the oral cavities of five healthy human volunteers for 72?h. The resulting biofilms were divided randomly between the following treatments: CAP (which varied in mean power, treatment duration, and/or the gas mixture), and untreated and treated controls (diode laser, air-abrasion, chlorhexidine). The viability, quantity, and morphology of the biofilms were determined by live/dead staining, inoculation onto blood agar, quantification of the total protein content, and scanning electron microscopy. Exposure to CAP significantly reduced the viability and quantity of biofilms compared with the positive control treatments. The efficacy of treatment with CAP correlated with the treatment duration and plasma power. No single method achieved complete biofilm removal; however, CAP may provide an effective support to established decontamination techniques for treatment of peri-implant diseases.     

Simultaneous ammonium-nitrogen and copper removal, and copper recovery using nitrifying biofilm from the ultra-compact biofilm reactor

Simultaneous ammonium-nitrogen (NH(4)(+)-N) and copper removal, and copper recovery in synthetic wastewater using nitrifying biofilm from an ultra-compact biofilm reactor (UCBR) was demonstrated in batch studies, which consisted of three phases: Phase 1 for NH(4)(+)-N and copper removals, Phase 2 for copper recovery, and Phase 3 for NH(4)(+)-N removal. The results showed that more than 96.3% of copper was removed within 60min, while 60.1% of the adsorbed copper was recovered through rinsing the biofilms with 0.1mM of ethylenediaminetetraacetic acid (EDTA). The nitrifying biofilm was able to adsorb 0.245mg of copper/g of biofilms. After recovery treatment, 29.4% of copper remained bound within the nitrifying biofilms. No significant inhibitory effects towards NH(4)(+)-N removal in the presence of 0.92mg copper/L was noted in Phase 1 compared with the control test. However, lower initial pH condition in the recovery process and the accumulation of copper on the biofilm led to 50% inhibition on NH(4)(+)-N removal efficiency in the subsequent phase.     

Profile of pterostilbene-induced redox homeostasis modulation in cardiac myoblasts and heart tissue

This study was designed to investigate the effect of pterostilbene (PTS) on cardiac oxidative stress in vitro, as this is a simple and promising methodology to study cardiac disease. Cardiac myoblasts (H9c2 cells) and homogenised cardiac tissue were incubated with the PTS and cyclodextrin (PTS?+?HPβCD) complex for 1 and 24 h, respectively, at concentrations of 50 μM for the cells and 25 and 50 μM for cardiac tissue. The PTS?+?HPβCD complex was used to increase the solubility of PTS in water. After the pretreatment period, cardiomyoblasts were challenged with hydrogen peroxide (6.67 μM) for 10?min, while cardiac tissue was submitted to a hydroxyl radical generator system (30?min). Cellular viability, oxidative stress biomarkers (e.g. total reactive oxygen species (ROS), carbonyl assay and lipoperoxidation) and the antioxidant response (e.g. sulfhydryl and the antioxidant enzyme activities of superoxide dismutase, catalase and glutathione peroxidase) were evaluated. In cardiomyoblasts, the PTS?+?HPβCD complex (50 μM) increased cellular viability. Moreover, the PTS?+?HPβCD complex also significantly increased sulfhydryl levels in the cells submitted to an oxidative challenge. In cardiac tissue, lipid peroxidation, carbonyls and ROS levels were significantly increased in the groups submitted to oxidative damage, while the PTS?+?HPβCD complex significantly reduced ROS levels in these groups. In addition, the PTS?+?HPβCD complex also provoked increased catalase activity in both experimental protocols. These data suggest that the PTS?+?HPβCD complex may play a cardioprotective role through a reduction of ROS levels associated with an improved antioxidant response.