scholarly journals Biofilm Formation and Methylene Blue-mediated Photodynamic Inactivation of Vibrio Parahaemolyticus in the Sea Food Industry

2020 ◽  
Vol 10 (3) ◽  
Author(s):  
Xiaoting Zhang ◽  
Qian Wu ◽  
Shuze Tang ◽  
William W Riley ◽  
Zhenqiang Chen
Keyword(s):  
Methylene Blue ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Vibrio Parahaemolyticus ◽  
Food Industry ◽  
Membrane Integrity ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Photodynamic Inactivation ◽  
Log Reduction

This study was conducted to better understand the mechanism of Vibrio Parahaemolyticus biofilm formation and to assess the inactivation effects of methylene blue-mediated photodynamic inactivation (PDI) technology as a preventative measure. Optical microscopy, following crystal violet staining, was used to observe the kinetics of V. parahaemolyticus biofilm formation. The crystal violet-based assay was performed in microtiter plates, and it was employed to determine which factors were most influential in the formation of the biofilms. Colony counting and confocal laser scanning microscopy (CLSM) were used to test the inactivation effect of methylene blue-mediated photodynamic technology on the biofilms. V. parahaemolyticus has the ability to form biofilms, as evidenced by their immediate adherence to glass surfaces and rapid maturity, within 24 h. High (7%) or low (0.5%) salinity was not conducive to the formation of biofilms, and rotational speed greater than 130 rpm also inhibited the process. A 4.05 log reduction in the concentration of viable biofilm cells was obtained with 100 μg/mL methylene blue and 20 min irradiation (24.996 J/cm2), but planktonic cells were more susceptible to the methylene blue-mediated photodynamic reaction (5.46 log reduction). The results presented here show that the methylene blue-mediated PDI technology is an effective means to inactivate V. parahaemolyticus by disrupting its membrane integrity and to inhibit the pathogen’s formation of protective biofilms. This technology is a valid tool that can be used to enhance food safety in the sea food industry.

scholarly journals Antibacterial activity and mechanism of sanguinarine against Providencia rettgeri in vitro

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9543
Author(s):  
Qian Zhang ◽  
Yansi Lyu ◽  
Jingkai Huang ◽  
Xiaodong Zhang ◽  
Na Yu ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Crystal Violet ◽  
Membrane Integrity ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Crystal Violet Staining ◽  
Providencia Rettgeri ◽  
Intracellular Atp ◽  
Atp Concentration

Background Sanguinarine (SAG), a benzophenanthridine alkaloid, occurs in Papaveraceas, Berberidaceae and Ranunculaceae families. Studies have found that SAG has antioxidant, anti-inflammatory, and antiproliferative activities in several malignancies and that it exhibits robust antibacterial activities. However, information reported on the action of SAG against Providencia rettgeri is limited in the literature. Therefore, the present study aimed to evaluate the antimicrobial and antibiofilm activities of SAG against P. rettgeri in vitro. Methods The agar dilution method was used to determine the minimum inhibitory concentration (MIC) of SAG against P. rettgeri. The intracellular ATP concentration, intracellular pH (pHin), and cell membrane integrity and potential were measured. Confocal laser scanning microscopy (CLSM), field emission scanning electron microscopy (FESEM), and crystal violet staining were used to measure the antibiofilm formation of SAG. Results The MIC of SAG against P. rettgeri was 7.8 μg/mL. SAG inhibited the growth of P. rettgeri and destroyed the integrity of P. rettgeri cell membrane, as reflected mainly through the decreases in the intracellular ATP concentration, pHin and cell membrane potential and significant changes in cellular morphology. The findings of CLSM, FESEM and crystal violet staining indicated that SAG exhibited strong inhibitory effects on the biofilm formation of P. rettgeri and led to the inactivity of biofilm-related P. rettgeri cells.

Characterization of Biofilm Formation by Cronobacter spp. Isolates of Different Food Origin under Model Conditions

2018 ◽  
Vol 82 (1) ◽  
pp. 65-77 ◽  
Author(s):  
MOHAMED A. ALY ◽  
ERIK REIMHULT ◽  
WOLFGANG KNEIFEL ◽  
KONRAD J. DOMIG
Keyword(s):  
Crystal Violet ◽  
Biofilm Formation ◽  
Sodium Acetate ◽  
Laser Scanning ◽  
Extracellular Polymeric Substances ◽  
Variable Temperature ◽  
Laser Scanning Microscopy ◽  
Individual Growth ◽  
Confocal Laser ◽  
Human Pathogens

ABSTRACT Cronobacter spp. are opportunistic human pathogens that cause serious diseases in neonates and immunocompromised people. Owing to their biofilm formation on various surfaces, both their detection and their removal from production plants constitute a major challenge. In this study, food samples were randomly collected in Austria and examined for the presence of Cronobacter spp. Presumptive isolates were identified by a polyphasic approach. Five percent of the samples were positive for C. sakazakii and 2.4% for C. dublinensis. Individual growth of the isolates was characterized based on lag time, growth rate, and generation time. During an incubation period of 6 to 72 h, biofilm formation of 11 selected isolates was quantified under model conditions by a crystal violet staining assay with 96-well plates with different carbon sources (lactose, glucose, maltose, sucrose, and sodium acetate) and NaCl levels and under variable temperature and pH conditions. Biofilm formation was more pronounced at lactose concentrations between 0.25 and 3% compared with 5% lactose, which lead to thinner layers. C. sakazakii isolate C7, isolated from infant milk powder, was the strongest biofilm producer at 10 mM Mg2+ and 5 mM Mn2+, 0.5% sodium acetate, at pH levels between 7 and 9 at 37°C for 24 h. C. sakazakii strain C6 isolated from a plant air filter was identified as a moderate biofilm former and C. sakazakii strain DSM 4485, a clinical isolate, as a weak biofilm former. Based on PCR detection, genes bcsA, bcsB, and bcsG encoding for cellulose could be identified as markers for biofilm formation. Isolates carrying bcsA and bcsB showed significantly stronger biofilm formation than isolates without these genes (P < 0.05), in strong correlation with the results obtained in the crystal violet assay. Further investigations using confocal laser scanning microscopy revealed that extracellular polymeric substances and glycocalyx secretions were the dominating components of the biofilms and that the viable fraction of bacteria in the biofilm decreased over time.

scholarly journals Quantifying Staphylococcus Aureus Biofilm Formation on Stainless Steel and Titanium Alloy Used in Orthopedic Fracture Fixation

2021 ◽  
Author(s):  
Shayesteh Beladi-Behbahani ◽  
Sarah M. Helms ◽  
John D. DesJardins ◽  
Marian S. Kennedy ◽  
Terri Bruce ◽  
...  
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Viable Cell ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Method

Abstract In order to assess and compare the antibacterial property of implants surfaces, a standard method is needed to quantify bacterial load. This study evaluated the effectiveness of three quantifying methods, namely, (I) crystal violet staining analysis, (II) ultrasound detachment with viable cell counts, and (III) confocal laser scanning microscopy for characterizing S. aureus Seattle 1945 (ATCC 25923) biofilm on metallic coupons. The accuracy of the results, time for completion, and ease of use of methods were compared. The crystal violet method is relatively faster and more straightforward for analyzing biofilm formation. However, the accuracy of the confocal laser scanning microscopy method is found to be considerably higher than that of the other methods. Confocal laser scanning microscopy method is considered to be more time-consuming for data collection and analysis and costlier. The ultrasound detachment followed by viable cell count of recovered cells is recommended for biofilm quantification analysis on orthopedic materials when there is a large number of samples (more than ten samples). This info could provide guidelines that would facilitate the selection of suitable method for quantifying biofilm formation on orthopedic implants based on investigators’ consideration on method accessibility, assay cost, assay time, and complexity of method.

Confocal and SEM imaging to demonstrate food pathogen- biofilm biocontrol by pyocyanin from Pseudomonas aeruginosa BTRY1

2016 ◽  
Vol 6 (01) ◽  
pp. 5218
Author(s):  
Laxmi Mohandas ◽  
Anju T. R. ◽  
Sarita G. Bhat*
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Antibiofilm Activity ◽  
Opportunistic Pathogens ◽  
Redox Active ◽  
Sem Imaging ◽  
The Impact

An assortment of redox-active phenazine compounds like pyocyanin with their characteristic blue-green colour are synthesized by Pseudomonas aeruginosa, Gram-negative opportunistic pathogens, which are also considered one of the most commercially valuable microorganisms. In this study, pyocyanin from Pseudomonas aeruginosa BTRY1 from food sample was assessed for its antibiofilm activity by micro titer plate assay against strong biofilm producers belonging to the genera Bacillus, Staphylococcus, Brevibacterium and Micrococcus. Pyocyanin inhibited biofilm activity in very minute concentrations. This was also confirmed by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM). Both SEM and CLSM helped to visualize the biocontrol of biofilm formation by eight pathogens. The imaging and quantification by CLSM also established the impact of pyocyanin on biofilm-biocontrol mainly in the food industry.

scholarly journals Bacterial lipopolysaccharides variably modulate in vitro biofilm formation of Candida species

2010 ◽  
Vol 59 (10) ◽  
pp. 1225-1234 ◽  
Author(s):  
H. M. H. N. Bandara ◽  
O. L. T. Lam ◽  
R. M. Watt ◽  
L. J. Jin ◽  
L. P. Samaranayake
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Significant Inhibition ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Bacterial Lipopolysaccharides ◽  
Species Specific ◽  
Biofilm Development

The objective of this study was to evaluate the effect of the bacterial endotoxin LPS on Candida biofilm formation in vitro. The effect of the LPS of Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens and Salmonella typhimurium on six different species of Candida, comprising Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA 646, was studied using a standard biofilm assay. The metabolic activity of in vitro Candida biofilms treated with LPS at 90 min, 24 h and 48 h was quantified by XTT reduction assay. Viable biofilm-forming cells were qualitatively analysed using confocal laser scanning microscopy (CLSM), while scanning electron microscopy (SEM) was employed to visualize the biofilm structure. Initially, adhesion of C. albicans was significantly stimulated by Pseudomonas and Klebsiella LPS. A significant inhibition of Candida adhesion was noted for the following combinations: C. glabrata with Pseudomonas LPS, C. tropicalis with Serratia LPS, and C. glabrata, C. parapsilosis or C. dubliniensis with Salmonella LPS (P<0.05). After 24 h of incubation, a significant stimulation of initial colonization was noted for the following combinations: C. albicans/C. glabrata with Klebsiella LPS, C. glabrata/C. tropicalis/C. krusei with Salmonella LPS. In contrast, a significant inhibition of biofilm formation was observed in C. glabrata/C. dubliniensis/C. krusei with Pseudomonas LPS, C. krusei with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. parapsilosis/C. dubliniensis /C. krusei with Salmonella LPS (P<0.05). On further incubation for 48 h, a significant enhancement of biofilm maturation was noted for the following combinations: C. glabrata/C. tropicalis with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. glabrata with Salmonella LPS, and a significant retardation was noted for C. parapsilosis/C. dubliniensis/C. krusei with Pseudomonas LPS, C. tropicalis with Serratia LPS, C. glabrata/C. parapsilosis/C. dubliniensis with Klebsiella LPS and C. dubliniensis with Salmonella LPS (P<0.05). These findings were confirmed by SEM and CLSM analyses. In general, the inhibition of the biofilm development of LPS-treated Candida spp. was accompanied by a scanty architecture with a reduced numbers of cells compared with the profuse and densely colonized control biofilms. These data are indicative that bacterial LPSs modulate in vitro Candida biofilm formation in a species-specific and time-dependent manner. The clinical and the biological relevance of these findings have yet to be explored.

scholarly journals Fluorescent Chitosan Nanogels Developed for Targeting Endothelial Cells of Axillary Lymph Nodes

2020 ◽  
Vol 4 (1) ◽  
pp. 12
Author(s):  
Miruna-Silvia Stan ◽  
Ionela Cristina Nica ◽  
Juliette Moreau ◽  
Maïté Callewaert ◽  
Cyril Cadiou ◽  
...  
Keyword(s):  
Membrane Damage ◽  
Membrane Integrity ◽  
Simian Virus 40 ◽  
Cancer Diagnostics ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microvascular Endothelial Cell ◽  
Axillary Lymph Nodes ◽  
Cell Periphery ◽  
Axillary Lymph

Nanogels are a novel class of three-dimensional cross-linked polymers able to retain high amounts of water in their network structure, with large potential applications in nanomedicine. In our study, the polymer matrix selected was chitosan, as this polysaccharide biopolymer composed of N-acetylglucosamine and glucosamine residues exhibits great biocompatibility and low toxicity. The preparation was performed by ionic gelation in the presence of hyaluronic acid and sodium tripolyphosphate, with rhodamine or fluorescein isothiocyanate molecules grafted on a chitosan backbone. In order to validate the possible usage of these chitosan-fluorophores conjugates for fluorescence imaging purposes in cancer diagnostics and therapy, their biological effect was assessed on SVEC4-10 cells (a simian virus 40-transformed mouse microvascular endothelial cell line). Cell viability, membrane integrity and nanogels uptake were examined following exposure for 6 and 24 h at concentrations up to 120 µg/mL. A good biocompatibility was obtained after both time intervals of incubation with nanogels, with no increase in cell death or membrane damage being noticed as compared to control. By examination on confocal laser scanning microscopy, both types of fluorescent nanogels agglomerated on the surface of the cell membrane, their cellular internalization being observed only for few cells, preferentially at the cell periphery. In conclusion, based on the biocompatibility of the nanogels, these can further incorporate gadolinium for an improved magnetic resonance imaging effect in nanomedicine.

scholarly journals Using micro-patterned surfaces to inhibit settlement and biofilm formation by Bacillus subtilis

2017 ◽  
Vol 63 (7) ◽  
pp. 608-620 ◽  
Author(s):  
Siyuan Chang ◽  
Xiaodong Chen ◽  
Shuo Jiang ◽  
Jinchun Chen ◽  
Lin Shi
Keyword(s):  
Bacillus Subtilis ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Patterned Surfaces ◽  
Heat Transfer Efficiency ◽  
Treated Sewage ◽  
Different Characteristics ◽  
Precision Balance

Biofilm is a biological complex caused by bacteria attachment to the substrates and their subsequent reproduction and secretion. This phenomenon reduces heat transfer efficiency and causes significant losses in treated sewage heat-recovering systems. This paper describes a physical approach to inhibit bacteria settlement and biofilm formation by Bacillus subtilis, which is the dominant species in treated sewage. Here, micro-patterned surfaces with different characteristics (stripe and cube) and dimensions (1–100 μm) were fabricated as surfaces of interest. Model sewage was prepared and a rotating coupon device was used to form the biofilms. Precision balance, scanning electron microscopy, and confocal laser scanning microscopy (CLSM) were employed to investigate the inhibitory effects and the mechanisms of the biofilm–surface interactions. The results have shown that surfaces with small pattern sizes (1 and 2 μm) all reduced biofilm formation significantly. Interestingly, the CLSM images showed that the surfaces do not play a role in “killing” the bacteria. These findings are useful for future development of new process surfaces on which bacteria settlement and biofilm formation can be inhibited or minimized.

scholarly journals Inhibition of Quorum Sensing and Biofilm Formation of Esculetin on Aeromonas Hydrophila

2021 ◽  
Vol 12 ◽  
Author(s):  
Bing Sun ◽  
Huaizhi Luo ◽  
Huan Jiang ◽  
Zhennan Wang ◽  
Aiqun Jia
Keyword(s):  
Quorum Sensing ◽  
Aeromonas Hydrophila ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Pcr Analysis ◽  
Swarming Motility ◽  
Gene Expression Levels ◽  
Good Agreement

Quorum sensing (QS) and biofilm formation inhibition activity of esculetin on Aeromonas hydrophila SHAe 115 were evaluated. Exposure to esculetin at 25, 50, and 100μg/ml significantly inhibited the production of protease and hemolysin, the formation of biofilms and attenuated the swarming motility of A. hydrophila SHAe 115. Biofilm forming inhibition was also observed through confocal laser scanning microscopy and scanning electron microscope. Quantitative real-time PCR analysis indicated that genes positively related to QS and biofilm formation were downregulated to varying degrees, while gene (litR) negatively related to biofilm formation was significantly upregulated. The phenotypic results were in good agreement with gene expression levels. These results indicated that esculetin would be a potential QS inhibitor for A. hydrophila.

scholarly journals Antifungal features and properties of chitosan/sandalwood oil Pickering emulsion coating stabilized by appropriate cellulose nanofiber dosage for fresh fruit application

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ata Aditya Wardana ◽  
Arisa Koga ◽  
Fumina Tanaka ◽  
Fumihiko Tanaka
Keyword(s):  
Laser Scanning ◽  
Cellulose Nanofibers ◽  
Membrane Integrity ◽  
Pickering Emulsion ◽  
Fresh Fruit ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Coating Film ◽  
Sandalwood Oil ◽  
Unstable Emulsion

AbstractA novel composite edible coating film was developed from 0.8% chitosan (CS) and 0.5% sandalwood oil (SEO). Cellulose nanofibers (CNFs) were used as a stabilizer agent of oil-in-water Pickering emulsion. We found four typical groups of CNF level-dependent emulsion stabilization, including (1) unstable emulsion in the absence of CNFs; (2) unstable emulsion (0.006–0.21% CNFs); (3) stable emulsion (0.24–0.31% CNFs); and (4) regular emulsion with the addition of surfactant. Confocal laser scanning microscopy was performed to reveal the characteristics of droplet diameter and morphology. Antifungal tests against Botrytis cinerea and Penicillium digitatum, between emulsion coating stabilized with CNFs (CS-SEOpick) and CS or CS-SEO was tested. The effective concentration of CNFs (0.24%) may improve the performance of CS coating and maintain CS-SEO antifungal activity synergistically confirmed with a series of assays (in vitro, in vivo, and membrane integrity changes). The incorporation of CNFs contributed to improve the functional properties of CS and SEO-loaded CS including light transmission at UV and visible light wavelengths and tensile strength. Atomic force microscopy and scanning electron microscopy were employed to characterize the biocompatibility of each coating film formulation. Emulsion-CNF stabilized coating may have potential applications for active coating for fresh fruit commodities.

Morphological and biochemical changes inPseudomonas fluorescensbiofilms induced by sub-inhibitory exposure to antimicrobial agents

2009 ◽  
Vol 55 (2) ◽  
pp. 163-178 ◽  
Author(s):  
James J. Dynes ◽  
John R. Lawrence ◽  
Darren R. Korber ◽  
George D.W. Swerhone ◽  
Gary G. Leppard ◽  
...  
Keyword(s):  
Antimicrobial Agent ◽  
Pseudomonas Fluorescens ◽  
Benzalkonium Chloride ◽  
Laser Scanning ◽  
Antimicrobial Agents ◽  
Membrane Integrity ◽  
Biochemical Changes ◽  
Laser Scanning Microscopy ◽  
Spectral Signature ◽  
Confocal Laser

Confocal laser scanning microscopy (CLSM) and scanning transmission X-ray microscopy (STXM) were used to examine the morphological and biochemical changes in Pseudomonas fluorescens biofilms grown in the presence of subinhibitory concentrations of 4 antimicrobial agents: triclosan, benzalkonium chloride, chlorhexidine dihydrochloride, and trisodium phosphate. CLSM analyses using the stains SYTO9 and propidium iodide indicated that the antimicrobial agents affected cell membrane integrity and cellular density to differing degrees. However, fluorescein diacetate assays and plate counts demonstrated that the cells remained metabolically active. Fluorescent lectin binding assays showed that changes in the arrangement and composition of the exopolymer matrix of the biofilms also occurred and that these changes depended on the antimicrobial agent. Detailed single cell analyses using STXM provided evidence that the cell morphology, and the spatial distribution and relative amounts of protein, lipids and polysaccharides in the biofilms and within the cells were different for each antimicrobial. The distribution of chlorhexidine in the biofilm, determined from its distinct spectral signature, was localized mainly inside the bacterial cells. Each antimicrobial agent elicited a unique response; P. fluorescens cells and biofilms changed their morphology and architecture, as well as the distribution and abundance of biomacromolecules, in particular the exopolymer matrix. Pseudomonas fluorescens also exhibited adaptation to benzalkonium chloride at 10 µg/mL. Our observations point to the importance of changes in the quantity and chemistry of the exopolymeric matrix in the response to antimicrobial agents and suggest their importance as targets for control.

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