Pseudomonas aeruginosa is one of the most important pathogenic bacteria related to biofilm infections. Due to the biofilm multi-drug resistance, methods of biofilm formation enumeration are of ...interest for assessment of efficient drug regimen development for biofilm inhibition or eradication. There are many different assay methods to determine the biofilm formation, using vital or non-vital dyes. The primary aim of the current study was to develop an assay using a member of tetrazolium salts family, 2,3,5-triphenyl-tetrazolium chloride (TTC), for detection of P. aeruginosa biofilm formation in 96-well microtiter plates and also a method of Minimum Biofilm Inhibitory Concentration (MBIC) determination of antibiotics against P. aeruginosa PAO1. Furthermore, the assay was optimized for TTC concentration, wavelength and period of incubation for 4 different antibiotics. The optimized condition was then compared with two other prevalent methods: the crystal violet (CV) assay and the 2,3-bis (2-methoxy-4-nitro-5-sulfophenly)-5-(phenylamino)carbonyl-2H-tetrazolium hydroxide (XTT) assay. In general, the optimized TTC assay (0.5% TTC, 6h of incubation and absorbance measurement at 405nm for biofilm assay and 1% TTC, 5h of incubation and absorbance measurement at 490nm for MBIC determination) distinguished between biofilms formed by different concentrations of bacteria and also was able to detect lower amounts of biofilm formed in contrast to the other two assay methods suggesting that TTC assay is more sensitive and also less expensive than other vital staining methods.
•Less amounts of biofilm formed were detectable by TTC assays.•The accuracy and repeatability of the results is more in TTC assay.•The assay is more sensitive and yet less expensive than other vital staining methods.
Biofilm formation by food-related bacteria and food-related pathogenesis are significant problems in the food industry. Even though much disinfection and mechanical procedure exist for removal of ...biofilms, they may fail to eliminate pre-established biofilms. cis-2 decenoic acid (CDA), an unsaturated fatty acid messenger produced by Pseudomonas aeruginosa, is reportedly capable of inducing the dispersion of established biofilms by multiple types of microorganisms. However, whether CDA has potential to boost the actions of certain antimicrobials is unknown. Here, the activity of CDA as an inducer of pre-established biofilms dispersal, formed by four main food pathogens; Staphylococcus aureus, Bacillus cereus, Salmonella enterica and E. coli, was measured using both semi-batch and continuous cultures bioassays. To assess the ability of CDA combined biocides treatments to remove pre-established biofilms formed on stainless steel discs, CFU counts were performed for both treated and untreated cultures. Eradication of the biofilms by CDA combined antibiotics was evaluated using crystal violet staining. The effect of CDA combined treatments (antibiotics and disinfectants) on biofilm surface area and bacteria viability was evaluated using fluorescence microscopy, digital image analysis and LIVE/DEAD staining. MICs were also determined to assess the probable inhibitory effects of CDA combined treatments on the growth of tested microorganisms' planktonic cells. Treatment of pre-established biofilms with only 310 nM CDA resulted in at least two-fold increase in the number of planktonic cells in all cultures. While antibiotics or disinfectants alone exerted a trivial effect on CFU counts and percentage of surface area covered by the biofilms, combinational treatments with both 310 nM CDA and antibiotics or disinfectants led to approximate 80% reduction in biofilm biomass. These data suggests that combined treatments with CDA would pave the way toward developing new strategies to control biofilms with widespread applications in industry as well as medicine.
The effects of treating two biopolymers (Trigonella foenum—graceum galactomannan and xanthan gum mixtures) with microwaves and ultrasound on the rheological aspects of O/W emulsions were ...investigated. The data obtained from steady shear flow were fitted with various models and the best were chosen due to the values of R2 and RMSE. The oscillatory shear rheology data demonstrated that the emulsions not treated with microwaves or ultrasound had viscous-like behavior and treated samples demonstrated weak gel behavior. The values obtained for various rheological parameters (especially apparent viscosity, storage modulus and loss modulus) indicated that fenugreek galactomannan had more impact on the rheological aspects of emulsions in comparison with xanthan gum. In addition, the synergistic interaction between two biopolymers, particularly in samples treated with ultrasound, resulted in better rheological aspects which could be affiliated with the strong bonds between the hydrocolloids. By treating the samples with microwaves and ultrasound, the emulsion stability values of the samples (especially those with a high ratio of galactomannan) significantly increased, which might be connected with various parameters, especially viscosity.
The catheterized urinary tract provides ideal conditions for the development of biofilm populations. Catheter-associated urinary tract infections (CAUTIs) are recalcitrant to existing antimicrobial ...treatments; therefore, established biofilms are not eradicated completely after treatment and surviving biofilm cells will carry on the infection. Cis-2-decenoic acid (CDA), an unsaturated fatty acid, is capable of inhibiting biofilm formation by Pseudomonas aeruginosa and of inducing the dispersion of established biofilms by multiple types of micro-organisms. Here, the ability of CDA to induce dispersal in pre-established single- and dual-species biofilms formed by Escherichia coli and Klebsiella pneumoniae was measured by using both semi-batch and continuous cultures bioassays. Removal of the biofilms by combined CDA and antibiotics (ciprofloxacin or ampicillin) was evaluated using microtitre plate assays (crystal violet staining). The c.f.u. counts were determined to assess the potential of combined CDA treatments to kill and eradicate pre-established biofilms formed on catheters. The effects of combined CDA treatments on biofilm surface area and bacteria viability were evaluated using fluorescence microscopy, digital image analysis and live/dead staining. To investigate the ability of CDA to prevent biofilm formation, single and mixed cultures were grown in the presence and absence of CDA. Treatment of pre-established biofilms with only 310 nM CDA resulted in at least threefold increase in the number of planktonic cells in all cultures tested. Whilst none of the antibiotics alone exerted a significant effect on c.f.u. counts and percentage of surface area covered by the biofilms, combined CDA treatments led to at least a 78% reduction in biofilm biomass in all cases. Moreover, most of the biofilm cells remaining on the surface were killed by antibiotics. The addition of 310 nM CDA significantly prevented biofilm formation by the tested micro-organisms, even within mixed cultures, indicating the ability of CDA to inhibit biofilm formation by other types of bacteria in addition to Pseudomonas aeruginosa. These findings suggested that the biofilm-preventive characteristics of CDA make it a noble candidate for inhibition of biofilm-associated infections such as CAUTIs, which paves the way toward developing new strategies to control biofilms in clinical as well as industrial settings.
First step of the pathway is catalyzed by ß-ketothiolase which combines two acetyl-CoA molecules to form acetoacetyl-CoA. ...polymerization of (R)-3-hydroxybutyryl-CoA monomers as the last step ...occurs in the presence of PHB synthase. Regions upstream of phbC gene in the form of GG-10N-GC, are promoter sequences which are recognized by a protein that is homologous of o54 in E. coli.11 Terminator sequence of phbC gene was found from WebGeSTer, the largest transcription terminator database (http://pallab.serc.iisc.ernet.in/gester).12 BamHI and HindIII restriction enzyme recognition sites were added to 5' end of forward and reverse cloning primers, respectively. ...a toehold with a length of 4 bp was placed at the 5' end of each cloning primer just behind restriction sites in order to optimize enzyme functions. ...the resulting amplified fragment included phbC gene, as well as 308 bp upstream and 92 bp downstream of the gene.
There is a lack of fundamental knowledge about the scale up of biosurfactant production. In order to develop suitable technology of commercialization, carrying out tests in shake flasks and ...bioreactors was essential. A reactor with integrated foam collector was designed for biosurfactant production using Bacillus subtilis isolated from agricultural soil. The yield of biosurfactant on biomass (Y p/x), biosurfactant on sucrose (Y p/s), and the volumetric production rate (Y) for shake flask were obtained about 0.45 g g⁻¹, 0.18 g g⁻¹, and 0.03 g l⁻¹ h⁻¹, respectively. The best condition for bioreactor was 300 rpm and 1.5 vvm, giving Y x/s, Y p/x, Y p/s, and Y of 0.42 g g⁻¹, 0.595 g g⁻¹, 0.25 g g⁻¹, and 0.057 g l⁻¹ h⁻¹, respectively. The biosurfactant maximum production, 2.5 g l⁻¹, was reached in 44 h of growth, which was 28% better than the shake flask. The obtained volumetric oxygen transfer coefficient (K L a) values at optimum conditions in the shake flask and the bioreactor were found to be around 0.01 and 0.0117 s⁻¹, respectively. Comparison of K L a values at optimum conditions shows that biosurfactant production scaling up from shake flask to bioreactor can be done with K L a as scale up criterion very accurately. Nearly 8% of original oil in place was recovered using this biosurfactant after water flooding in the sand pack.
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disorder in which lymphocytic infiltration mediated mainly by pro-inflammatory cytokines. In this study, we examined the effect of ...combined exercise training on the levels of IFN-γ, IL-4 and IL-17 in the plasma and the supernatant of peripheral blood lymphocytes in women with multiple sclerosis. Expanded Disability Status Scale (EDSS), VO
2max, muscle strength, and balance tests were obtained at baseline and post-treatment follow-up. Combined exercises training was designed for 24 sessions during 8
weeks. Each session was started with 5
min warm-up and was followed by 10
min stretch training, 20
min aerobic exercises and 20
min resistance–endurance training. The disability score was significantly decreased in test MS subjects after 8
weeks combined exercise training. Muscle strength and balance were increased significantly after the training program in test group. In this study, plasma, and peripheral blood mononuclear cell (PBMC) IL-17 and IFN-γ production was significantly decreased after 8
weeks combined training. Our findings suggest that combined training has useful anti-inflammatory effects by decrease in PBMC and plasma IL-17 production.
► Combined exercise training has anti-inflammatory effect on MS patients. ► Serum level of IL-17 and IFN-γ was decreased after combined exercise training. ► PBMC production of IL-17 and IFN-γ was decreased after combined exercise training. ► Combined exercise training improves the Physical activity and EDSS of MS subjects.
•Production of schizophyllan from date syrup with excellent rheological properties.•Effect of recovery methods on purity and molecular structure was characterized.•Relationship of purity and ...structural features with the rheological properties was elucidated.
This study investigates the effects of different recovery procedures on high molar mass schizophyllan produced by Schizophyllum commune using low value agricultural residues. Recovered extracellular polysaccharides (EPSs) were compared in terms of purity, sugar composition, degree of branching, molecular weight, and rheological properties. Performing different recovery methods, such as re-dissolving in water and re-precipitation with ethanol on produced EPS, provided schizophyllan with purity similar to the commercial grade. Besides, Freeze-thawing cycles allowed the fractionation of schizophyllan based on branching degree and solubility. The EPSs with higher purity and lower degree of branching (less conformational flexibility) showed higher viscosity. This study evidences the possibility of producing EPSs with excellent rheological properties using low value agricultural side products. Furthermore, our results demonstrate the importance of recovery methods for tailoring the purity, molecular structure and macroscopic properties of the produced polysaccharides for specific applications.
In this study, the efficiency improvement of three moving bed biofilm reactors (MBBRs) was investigated by inoculation of activated sludge cells (R1), mixed culture of eight strong phenol-degrading ...bacteria consisted of
Pseudomonas
spp. and
Acinetobacter
spp. (R2) and the combination of both (R3). Biofilm formation ability of eight bacteria was assessed initially using different methods and media. Maximum degradation of phenol, COD, biomass growth and also changes in organic loading shock were used as parameters to measure the performance of reactors. According to the results, all eight strains were determined as enhanced biofilm forming bacteria (EBFB). Under optimum operating conditions, more than 90% of initial COD load of 2795 mg L
−1
was reduced at 24 HRT in R3 while this reduction efficiency was observed in concentrations of 1290 mg L
−1
and 1935 mg L
−1
, in R1 and R2, respectively. When encountering phenol loading shock—twice greater than optimum amount-R1, R2 and R3 managed to return to the steady-state condition within 32, 24 and 18 days, respectively. SEM microscopy and biomass growth measurements confirmed the contribution of more cells to biofilm formation in R3 followed by R2. Additionally, established biofilm in R3 was more resistant to phenol loading shock which can be attributed to the enhancer role of EBFB strains in this reactor. It has been demonstrated that the bacteria with both biofilm-forming and contaminant-degrading abilities are not only able to promote the immobilization of other favorable activated sludge cells in biofilm structure, but also cooperate in contaminant degradation which all consequently lead to improvement of treatment efficiency.
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