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.

scholarly journals Comparison of the Antimicrobial Effects of Chlorine, Silver Ion, and Tobramycin on Biofilm

2008 ◽  
Vol 52 (4) ◽  
pp. 1446-1453 ◽  
Author(s):  
Jaeeun Kim ◽  
Betsey Pitts ◽  
Philip S. Stewart ◽  
Anne Camper ◽  
Jeyong Yoon
Keyword(s):  
Laser Scanning ◽  
Antimicrobial Agents ◽  
Respiratory Activity ◽  
Membrane Integrity ◽  
Laser Scanning Microscopy ◽  
Epifluorescence Microscopy ◽  
Confocal Laser ◽  
Tetrazolium Chloride ◽  
Antimicrobial Efficiency ◽  
Sequential Treatments

ABSTRACT The systematic understanding of how various antimicrobial agents are involved in controlling biofilms is essential in order to establish an effective strategy for biofilm control, since many antimicrobial agents are effective against planktonic cells but are ineffective when they are used against the same bacteria growing in a biofilm state. Three different antimicrobial agents (chlorine, silver, and tobramycin) and three different methods for the measurement of membrane integrity (plate counts, the measurement of respiratory activity with 5-cyano-2,3-ditolyl tetrazolium chloride [CTC] staining, and BacLight Live/Dead staining) were used along with confocal laser scanning microscopy (CLSM) and epifluorescence microscopy to examine the activities of the antimicrobials on biofilms in a comparative way. The three methods of determining the activities of the antimicrobials gave very different results for each antimicrobial agent. Among the three antimicrobials, tobramycin appeared to be the most effective in reducing the respiratory activity of biofilm cells, based upon CTC staining. In contrast, tobramycin-treated biofilm cells maintained their membrane integrity better than chlorine- or silver-treated ones, as evidenced by imaging by both CLSM and epifluorescence microscopy. Combined and sequential treatments with silver and tobramycin showed an enhanced antimicrobial efficiency of more than 200%, while the antimicrobial activity of either chlorine or tobramycin was antagonized when the agents were used in combination. This observation makes sense when the different oxidative reactivities of chlorine, silver, and tobramycin are considered.

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.

scholarly journals Effect of Sodium Hypochlorite and Benzalkonium Chloride on the Structural Parameters of the Biofilms Formed by Ten Salmonella enterica Serotypes

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 154 ◽  
Author(s):  
Rosa Capita ◽  
Silvia Fernández-Pérez ◽  
Laura Buzón-Durán ◽  
Carlos Alonso-Calleja
Keyword(s):  
Sodium Hypochlorite ◽  
Salmonella Enterica ◽  
Benzalkonium Chloride ◽  
Laser Scanning ◽  
Surface Coverage ◽  
Structural Parameters ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Low Concentrations ◽  
Practical Field

The influence of the strain on the ability of Salmonella enterica to form biofilms on polystyrene was investigated by confocal laser scanning microscopy. The effects of sodium hypochlorite with 10% active chlorine (SHY; 25,000, 50,000, or 100,000 ppm), and benzalkonium chloride (BZK; 1000, 5000, or 10,000 ppm) on twenty-four-hour-old biofilms was also determined. The biofilms of ten Salmonella enterica isolates from poultry (S. Agona, S. Anatum, S. Enteritidis, S. Hadar, S. Infantis, S. Kentucky, S. Thompson, S. Typhimurium, monophasic variant of S. Typhimurium 1,4,(5),12:i:-, and S. Virchow) were studied. Biofilms produced by S. Anatum, S. Hadar, S. Kentucky, and S. Typhimurium showed a trend to have the largest biovolume and the greatest surface coverage and thickness. The smallest biofilms (P < 0.01) in the observation field (14.2 × 103 µm2) were produced by S. Enteritidis and S. 1,4,(5),12:i:- (average 12.9 × 103 ± 9.3 × 103 µm3) compared to the rest of the serotypes (44.4 × 103 ± 24.7 × 103 µm3). Biovolume and surface coverage decreased after exposure for ten minutes to SHY at 50,000 or 100,000 ppm and to BZK at 5000 or 10,000 ppm. However, the lowest concentrations of disinfectants increased biovolume and surface coverage in biofilms of several strains (markedly so in the case of BZK). The results from this study suggest that the use of biocides at low concentrations could represent a public health risk. Further research studies under practical field conditions should be appropriate to confirm these findings.

scholarly journals A Novel Hybrid Peptide Composed of LfcinB6 and KR-12-a4 With Enhanced Antimicrobial, Anti-Inflammatory and Anti-Biofilm Activities

2021 ◽  
Author(s):  
Chelladurai Ajish ◽  
Sungtae Yang ◽  
S. Dinesh Kumar ◽  
Eun Young Kim ◽  
Hye Jung Min ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Laser Scanning ◽  
Antimicrobial Agents ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Hybrid Peptide ◽  
Cell Selectivity ◽  
Anti Inflammatory

Abstract Hybridizing two known antimicrobial peptides (AMPs) is a simple and effective strategy for designing antimicrobial agents with enhanced cell selectivity against bacterial cells. Here, we generated a hybrid peptide Lf-KR in which LfcinB6 and KR-12-a4 were linked with a Pro hinge to obtain a novel AMP with potent antimicrobial, anti-inflammatory, and anti-biofilm activities. Lf-KR exerted superior cell selectivity for bacterial cells over sheep red blood cells. Lf-KR showed broad-spectrum antimicrobial activities (MIC: 4–8 mM) against tested 12 bacterial strains and retained its antimicrobial activity in the presence of salts at physiological concentrations. Membrane depolarization and dye leakage assays showed that the enhanced antimicrobial activity of Lf-KR was due to increased permeabilization and depolarization of microbial membranes. Lf-KR significantly inhibited the expression and production of pro-inflammatory cytokines (NO and TNF-a) in LPS-stimulated mouse macrophage RAW264.7 cells. In addition, Lf-KR showed a powerful eradication effect on preformed multidrug-resistant Pseudomonas aeruginosa (MDRPA) biofilms. We confirmed using confocal laser scanning microscopy that a large portion of the preformed MDRPA biofilm structure was perturbed by the addition of Lf-KR. Collectively, our results suggest that Lf-KR can be an antimicrobial, anti-inflammatory, and anti-biofilm candidate as a pharmaceutical agent.

scholarly journals Suppression of Xanthomonas oryzae pv. oryzae biofilm formation by Acacia mangium methanol leaf extract

2020 ◽  
Author(s):  
S. N. Sarah Shafiei ◽  
K. Ahmad ◽  
N. F. M. Ikhsan ◽  
S. I. Ismail ◽  
K. Sijam
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Leaf Extract ◽  
Antimicrobial Agents ◽  
Acacia Mangium ◽  
Positive Control ◽  
Laser Scanning Microscopy ◽  
Xanthomonas Oryzae ◽  
Confocal Laser ◽  
Biofilm Inhibition

Abstract Xanthomonas oryzae pv. oryzae (Xoo), a pathogen responsible for rice bacterial leaf blight, produces biofilm to protect viable Xoo cells from antimicrobial agents. A study was conducted to determine the potency of Acacia mangium methanol (AMMH) leaf extract as a Xoo biofilm inhibitor. Four concentrations (3.13, 6.25, 9.38, and 12.5 mg/mL) of AMMH leaf extract were tested for their ability to inhibit Xoo biofilm formation on a 96-well microtiter plate. The results showed that the negative controls had the highest O.D. values from other treatments, indicating the intense formation of biofilm. This was followed by the positive control (Streptomycin sulfate, 0.2 mg/mL) and AMMH leaf extract at concentration 3.13 mg/mL, which showed no significant differences in their O.D. values (1.96 and 1.57, respectively). All other treatments at concentrations of 6.25, 9.38, and 12.5 mg/mL showed no significant differences in their O.D. values (0.91, 0.79, and 0.53, respectively). For inhibition percentages, treatment with concentration 12.5 mg/mL gave the highest result (81.25%) followed by treatment at concentrations 6.25 and 9.38 mg/mL that showed no significant differences in their inhibition percentage (67.75% and 72.23%, respectively). Concentration 3.13 mg/mL resulted in 44.49% of biofilm inhibition and the positive control resulted in 30.75% of biofilm inhibition. Confocal laser scanning microscopy (CLSM) analysis of Xoo biofilm inhibition and breakdown showed the presence of non-viable Xoo cells and changes in aggregation size due to increase in AMMH leaf extract concentration. Control slides showed the absence of Xoo dead cells.

scholarly journals Oxidative Imbalance in Candida tropicalis Biofilms and Its Relation With Persister Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
María A. da Silva ◽  
José L. Baronetti ◽  
Paulina L. Páez ◽  
María G. Paraje
Keyword(s):  
Candida Tropicalis ◽  
Laser Scanning ◽  
Structural Changes ◽  
Antimicrobial Agents ◽  
Laser Scanning Microscopy ◽  
Yeast Cells ◽  
Confocal Laser ◽  
Antioxidant Systems ◽  
Roughness Coefficient ◽  
Persister Cells

BackgroundPersister cells (PCs) make up a small fraction of microbial population, can survive lethal concentrations of antimicrobial agents. In recent years, Candida tropicalis has emerged as being a frequent fungal agent of medical devices subject to biofilm infections. However, PCs are still poorly understood.ObjectivesThis study aimed to investigate the relation of PCs on the redox status in C. tropicalis biofilms exposed to high doses of Amphotericin B (AmB), and alterations in surface topography and the architecture of biofilms.MethodsWe used an experimental model of two different C. tropicalis biofilms exposed to AmB at supra minimum inhibitory concentration (SMIC80), and the intra- and extracellular reactive oxygen species (iROS and eROS), reactive nitrogen species (RNS) and oxidative stress response were studied. Light microscopy (LM) and confocal laser scanning microscopy (CLSM) were also used in conjunction with the image analysis software COMSTAT.ResultsWe demonstrated that biofilms derived from the PC fraction (B2) showed a higher capacity to respond to the stress generated upon AmB treatment, compared with biofilms obtained from planktonic cells. In B2, a lower ROS and RNS accumulation was observed in concordance with higher activation of the antioxidant systems, resulting in an oxidative imbalance of a smaller magnitude compared to B1. LM analysis revealed that the AmB treatment provoked a marked decrease of biomass, showing a loss of cellular aggrupation, with the presence of mostly yeast cells. Moreover, significant structural changes in the biofilm architecture were noted between both biofilms by CLSM—COMSTAT analysis. For B1, the quantitative parameters bio-volume, average micro-colony volume, surface to bio-volume ratio and surface coverage showed reductions upon AmB treatment, whereas increases were observed in roughness coefficient and average diffusion distance. In addition, untreated B2 was substantially smaller than B1, with less biomass and thickness values. The analysis of the above-mentioned parameters also showed changes in B2 upon AmB exposure.ConclusionTo our knowledge, this is the first study that has attempted to correlate PCs of Candida biofilms with alterations in the prooxidant-antioxidant balance and the architecture of the biofilms. The finding of regular and PCs with different cellular stress status may help to solve the puzzle of biofilm resistance, with redox imbalance possibly being an important factor.

scholarly journals Effect of Cinnamon Oil on icaA Expression and Biofilm Formation by Staphylococcus epidermidis

2009 ◽  
Vol 75 (21) ◽  
pp. 6850-6855 ◽  
Author(s):  
Titik Nuryastuti ◽  
Henny C. van der Mei ◽  
Henk J. Busscher ◽  
Susi Iravati ◽  
Abu T. Aman ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Antimicrobial Agents ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Pcr Analysis ◽  
Minimal Bactericidal Concentration ◽  
Cinnamon Oil ◽  
Broth Microdilution Method

ABSTRACT Staphylococcus epidermidis is notorious for its biofilm formation on medical devices, and novel approaches to prevent and kill S. epidermidis biofilms are desired. In this study, the effect of cinnamon oil on planktonic and biofilm cultures of clinical S. epidermidis isolates was evaluated. Initially, susceptibility to cinnamon oil in planktonic cultures was compared to the commonly used antimicrobial agents chlorhexidine, triclosan, and gentamicin. The MIC of cinnamon oil, defined as the lowest concentration able to inhibit visible microbial growth, and the minimal bactericidal concentration, the lowest concentration required to kill 99.9% of the bacteria, were determined using the broth microdilution method and plating on agar. A checkerboard assay was used to evaluate the possible synergy between cinnamon oil and the other antimicrobial agents. The effect of cinnamon oil on biofilm growth was studied in 96-well plates and with confocal laser-scanning microscopy (CLSM). Biofilm susceptibility was determined using a metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Real-time PCR analysis was performed to determine the effect of sub-MIC concentrations of cinnamon oil on expression of the biofilm-related gene, icaA. Cinnamon oil showed antimicrobial activity against both planktonic and biofilm cultures of clinical S. epidermidis strains. There was only a small difference between planktonic and biofilm MICs, ranging from 0.5 to 1% and 1 to 2%, respectively. CLSM images indicated that cinnamon oil is able to detach and kill existing biofilms. Thus, cinnamon oil is an effective antimicrobial agent to combat S. epidermidis biofilms.

Comparison of Efficacy of Povidone-Iodine, Ethanol, and an Aerosol Formulation of Silver Sulfadiazine in Controlling Microbial Burden on Sutures From Clean Surgeries

2021 ◽  
Author(s):  
Gustavo E Prezzavento ◽  
Carlos R Mas ◽  
Juan Achaval Rodríguez ◽  
Rodrigo N Juárez Calvi ◽  
Laura Piskulic ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Microbial Contamination ◽  
Antimicrobial Agents ◽  
Povidone Iodine ◽  
Silver Sulfadiazine ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microbial Cells ◽  
Surgical Suture ◽  
Biofilm Development

Abstract In the surgical suture, the implanted thread can be a source of microbial contamination. Implanted materials are frequently described as being substrates prone for biofilm development provoking surgical site infections. Treatment of postsurgical wounds with different topical antimicrobial agents is a current practice applied to every patient. However, to date, there is little evidence on the efficacy of different antiseptic treatments on suture materials in preventing environmental or skin bacterial adhesion and further infection. Here, the authors compared the ability of an aerosol formulation of silver sulfadiazine, vitamin A, and lidocaine (AF-SSD) and of two of the most frequently used topical treatments, povidone-iodine and ethanol, in eradicating or controlling the microbial contamination of suture threads in patients who have undergone clean surgeries. Postsurgical suture threads treated with AF-SSD showed a significantly reduced proportion of contaminated samples containing viable microbial cells compared with those treated with povidone-iodine or ethanol. Furthermore, those samples that were positive for bacterial growth showed a lesser number of viable cells in AF-SSD-treated sutures than those treated with povidone-iodine or ethanol. Confocal laser scanning microscopy showed that AF-SSD-treated postsurgical sutures presented significantly less attached microbial cells than povidone-iodine and ethanol, with scarce observable microbial cells on the surface of the suture. Taken together, the results suggest that treatment with AF-SSD is more effective than the other two antiseptics, and there is a potential for improvement in reducing the microbial burden of implanted materials such as the suture thread.

scholarly journals Antifungal Features and Properties of Chitosan/Sandalwood Oil Pickering Emulsion Coating Stabilized by Appropriate Cellulose Nanofiber Dosage for Fresh Fruit Application

2021 ◽  
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

Abstract A novel composite edible coating film was developed from 0.8% chitosan (CS) and 0.5% n 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.

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