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.

Interactions of WPI and Xanthan in Microstructure and Rheological Properties of Gels and Emulsions

2007 ◽  
Vol 3 (4) ◽  
Author(s):  
Kooshan Nayebzadeh ◽  
Jianshe Chen ◽  
SM Mohammad Mousavi
Keyword(s):  
Phase Separation ◽  
Whey Protein ◽  
Viscoelastic Properties ◽  
Xanthan Gum ◽  
Laser Scanning ◽  
Structural Changes ◽  
Spherical Shape ◽  
Laser Scanning Microscopy ◽  
Concentration Addition ◽  
Confocal Laser

The effect of addition of xanthan gum (0.05, 0.1, 0.15, 0.25% weight/volume) on the formation and rheology of whey protein isolate (WPI)-xanthan gum gels has been investigated at neutral pH. The elastic modulus (G') values of the gelling test were compared. Low concentration of xanthan added (<0.05%,w/v) has a synergistic effect on the gel strength depend on phase separation, so that whey proteins concentrated in their phase and finally mixed gels with xanthan would be stronger than WPI gels. At higher xanthan concentration (> 0.05%, w/v), antagonist effect was observed by reducing the connection between clusters of whey protein by xanthan, so aggregation disruption and a related decrease in (G'). The phase separation microstructure of WPI-stabilized emulsion containing xanthan gum added has been investigated by rheology and confocal laser scanning microscopy. Xanthan was stained with Fluorescein 5(6)-isothiocyanate (FITC). Low xanthan concentration addition lead to depletion flocculation and increasing the xanthan concentration cause to increase the viscoelasticity of aqueous phase, so retarded macroscopic phase separation over period investigated. Structural changes in emulsion were observed in viscoelastic properties of separated phase in the rheometer. The CLSM image shows different phase which have different viscoelastic properties; xanthan-rich region transforms into the spherical shape which has the lowest interfacial energy and gradually two separated ultimately.

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 The Chromosomal Toxin Gene yafQ Is a Determinant of Multidrug Tolerance for Escherichia coli Growing in a Biofilm

2009 ◽  
Vol 53 (6) ◽  
pp. 2253-2258 ◽  
Author(s):  
Joe J. Harrison ◽  
William D. Wade ◽  
Sarah Akierman ◽  
Caterina Vacchi-Suzzi ◽  
Carol A. Stremick ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Cell Survival ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Cell Counting ◽  
Confocal Laser ◽  
Toxin Gene ◽  
Persister Cells ◽  
E Coli

ABSTRACT Escherichia coli is refractory to elevated doses of antibiotics when it is growing in a biofilm, and this is potentially due to high numbers of multidrug-tolerant persister cells in the surface-adherent population. Previously, the chromosomal toxin-antitoxin loci hipBA and relBE have been linked to the frequency at which persister cells occur in E. coli populations. In the present study, we focused on the dinJ-yafQ-encoded toxin-antitoxin system and hypothesized that deletion of the toxin gene yafQ might influence cell survival in antibiotic-exposed biofilms. By using confocal laser scanning microscopy and viable cell counting, it was determined that a ΔyafQ mutant produced biofilms with a structure and a cell density equivalent to those of the parental strain. In-depth susceptibility testing identified that relative to wild-type E. coli, the ΔyafQ strain had up to a ∼2,400-fold decrease in cell survival after the biofilms were exposed to bactericidal concentrations of cefazolin or tobramycin. Corresponding to these data, controlled overexpression of yafQ from a high-copy-number plasmid resulted in up to a ∼10,000-fold increase in the number of biofilm cells surviving exposure to these bactericidal drugs. In contrast, neither the inactivation nor the overexpression of yafQ affected the tolerance of biofilms to doxycycline or rifampin (rifampicin). Furthermore, deletion of yafQ did not affect the tolerance of stationary-phase planktonic cells to any of the antibacterials tested. These results suggest that yafQ mediates the tolerance of E. coli biofilms to multiple but specific antibiotics; moreover, our data imply that this cellular pathway for persistence is likely different from that of multidrug-tolerant cells in stationary-phase planktonic cell cultures.

Interspecies Interactions in Dual-Species Biofilms Formed by Psychrotrophic Bacteria and the Tolerance of Sessile Communities to Disinfectants

2020 ◽  
Vol 83 (6) ◽  
pp. 951-958 ◽  
Author(s):  
LEI YUAN ◽  
NI WANG ◽  
FAIZAN A. SADIQ ◽  
GUOQING HE
Keyword(s):  
Hydrogen Peroxide ◽  
Sodium Hypochlorite ◽  
Peracetic Acid ◽  
Laser Scanning ◽  
Structural Changes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Interspecies Interactions ◽  
Mixed Species ◽  
Psychrotrophic Bacteria

ABSTRACT Biofilms on the surface of food processing equipment act as potential reservoirs of microbial contamination. Bacterial interactions are believed to play key roles in both biofilm formation and antimicrobial tolerance. In this study, Aeromonas hydrophila, Chryseobacterium oncorhynchi, and Pseudomonas libanensis, which were previously isolated from Chinese raw milk samples, were selected to establish two dual-species biofilm models (P. libanensis plus A. hydrophila and P. libanensis plus C. oncorhynchi) on stainless steel at 7°C. Subsequently, three disinfectants, hydrogen peroxide (100 ppm), peracetic acid (100 ppm), and sodium hypochlorite (100 ppm), were used to treat the developed sessile communities for 10 min. Structural changes after exposure to disinfectants were analyzed with confocal laser scanning microscopy. The cell numbers of both A. hydrophila and C. oncorhynchi recovered from surfaces increased when grown as dual species biofilms with P. libanensis. Dual-species biofilms were more tolerant of disinfectants than were each single-species biofilm. Peracetic acid was the most effective disinfectant for removing biofilms, followed by hydrogen peroxide and sodium hypochlorite. The results expand the knowledge of mixed-species biofilms formed by psychrotrophic bacteria and will be helpful for developing effective strategies to eliminate bacterial mixed-species biofilms. HIGHLIGHTS

scholarly journals Metabolic Differentiation in Biofilms as Indicated by Carbon Dioxide Production Rates

2009 ◽  
Vol 76 (4) ◽  
pp. 1189-1197 ◽  
Author(s):  
Elanna Bester ◽  
Otini Kroukamp ◽  
Gideon M. Wolfaardt ◽  
Leandro Boonzaaier ◽  
Steven N. Liss
Keyword(s):  
Carbon Dioxide ◽  
Laser Scanning ◽  
Extracellular Polymeric Substances ◽  
Carbon Dioxide Production ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Roughness Coefficient ◽  
Production Rates ◽  
Mechanical Methods ◽  
Biofilm Development

ABSTRACT The measurement of carbon dioxide production rates as an indication of metabolic activity was applied to study biofilm development and response of Pseudomonas sp. biofilms to an environmental disturbance in the form of a moving air-liquid interface (i.e., shear). A differential response in biofilm cohesiveness was observed after bubble perturbation, and the biofilm layers were operationally defined as either shear-susceptible or non-shear-susceptible. Confocal laser scanning microscopy and image analysis showed a significant reduction in biofilm thickness and biomass after the removal of the shear-susceptible biofilm layer, as well as notable changes in the roughness coefficient and surface-to-biovolume ratio. These changes were accompanied by a 72% reduction of whole-biofilm CO2 production; however, the non-shear-susceptible region of the biofilm responded rapidly after the removal of the overlying cells and extracellular polymeric substances (EPS) along with the associated changes in nutrient and O2 flux, with CO2 production rates returning to preperturbation levels within 24 h. The adaptable nature and the ability of bacteria to respond to environmental conditions were further demonstrated by the outer shear-susceptible region of the biofilm; the average CO2 production rate of cells from this region increased within 0.25 h from 9.45 ± 5.40 fmol of CO2·cell−1·h−1 to 22.6 ± 7.58 fmol of CO2·cell−1·h−1 when cells were removed from the biofilm and maintained in suspension without an additional nutrient supply. These results also demonstrate the need for sufficient monitoring of biofilm recovery at the solid substratum if mechanical methods are used for biofouling control.

scholarly journals Individual or Combined Effects of Meropenem, Imipenem, Sulbactam, Colistin, and Tigecycline on Biofilm-Embedded Acinetobacter baumannii and Biofilm Architecture

2016 ◽  
Vol 60 (8) ◽  
pp. 4670-4676 ◽  
Author(s):  
Yung-Chih Wang ◽  
Shu-Chen Kuo ◽  
Ya-Sung Yang ◽  
Yi-Tzu Lee ◽  
Chun-Hsiang Chiu ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Laser Scanning ◽  
Bactericidal Effect ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Roughness Coefficient ◽  
Content Type ◽  
Biofilm Thickness ◽  
Carbapenem Resistant ◽  
Biofilm Architecture

ABSTRACTAcinetobacter baumanniibiofilms are difficult to eradicate. We investigated the effects of meropenem (2 mg/liter), imipenem (2 mg/liter), sulbactam (4 mg/liter), colistin (2 mg/liter), and tigecycline (2 mg/liter), alone or in combination, on biofilm-embedded carbapenem-resistant and carbapenem-susceptibleA. baumannii(CRAb and CSAb, respectively) cells, as well as on the architecture of the biofilms.A. baumanniiATCC 15151 (Ab15151) and its OXA-82-overproducing transformant, along with two clinical CSAb and two clinical CRAb isolates of differing clonalities, were used. The minimal bactericidal concentrations for biofilm-embedded cells of the six tested isolates were >50-fold those of their planktonic cells. When used individually, meropenem exhibited a higher killing effect than the other four antimicrobials on biofilm-embedded CSAb cells in the colony biofilm assay. For two clinical CRAb isolates, meropenem plus sulbactam or sulbactam plus tigecycline showed >100-fold the bactericidal effect exhibited by these agents used alone after 48 h of treatment. The effect of antimicrobials on the architecture of Ab15151 biofilm emitting green fluorescence was determined by confocal laser scanning microscopy using COMSTAT software. Significant decreases in the maximum biofilm thickness were observed after exposure to meropenem and imipenem. Meropenem plus sulbactam significantly decreased the biomass and mean thickness and increased the roughness coefficient of biofilms, but sulbactam plus tigecycline only decreased the maximum and mean biofilm thickness compared to any of these agents used alone. Meropenem was active against biofilm-embedded CSAb, whereas meropenem plus sulbactam exhibited synergism against biofilm-embedded CRAb and caused significantly more damage to the biofilm architecture than did any of the agents used alone.

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 Stay in Touch—The Cortical ER of Moss Protonemata in Osmotic Stress Situations

Plants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 421 ◽  
Author(s):  
Dominik Harant ◽  
Ingeborg Lang
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Laser Scanning ◽  
Structural Changes ◽  
Physcomitrella Patens ◽  
Dynamic Network ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Natural State ◽  
Cell Cortex

Plasmolysis is usually introduced to cell biology students as a tool to illustrate the plasma membrane: hypertonic solutions cause the living protoplast to shrink by osmotic water loss; hence, it detaches from the surrounding cell wall. What happens, however, with the subcellular structures in the cell cortex during this process of turgor loss? Here, we investigated the cortical endoplasmic reticulum (ER) in moss protonema cells of Physcomitrella patens in a cell line carrying a transgenic ER marker (GFP-HDEL). The plasma membrane was labelled simultaneously with the fluorescent dye FM4-64 to achieve structural separation. By placing the protonemata in a hypertonic mannitol solution (0.8 M), we were able to follow the behaviour of the cortical ER and the protoplast during plasmolysis by confocal laser scanning microscopy (CLSM). The protoplast shape and structural changes of the ER were further examined after depolymerisation of actin microfilaments with latrunculin B (1 µM). In its natural state, the cortical ER is a dynamic network of fine tubes and cisternae underneath the plasma membrane. Under acute and long-term plasmolysis (up to 45 min), changes in the protoplast form and the cortical ER, as well as the formation of Hechtian strands and Hechtian reticula, were observed. The processing of the high-resolution z-scans allowed the creation of 3D models and gave detailed insight into the ER of living protonema cells before, during and after plasmolysis.

scholarly journals Effect of Clove and Thyme Essential Oils on Candida Biofilm Formation and the Oil Distribution in Yeast Cells

Molecules ◽  
2019 ◽  
Vol 24 (10) ◽  
pp. 1954 ◽  
Author(s):  
Katarzyna Rajkowska ◽  
Paulina Nowicka-Krawczyk ◽  
Alina Kunicka-Styczyńska
Keyword(s):  
Essential Oils ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Yeast Cells ◽  
Confocal Laser ◽  
Minimal Inhibitory Concentrations ◽  
Candida Sp ◽  
A Cell ◽  
Candida Biofilm

Candida biofilm structure is particularly difficult to eradicate, since biofilm is much more resistant to antifungal agents than planktonic cells. In this context, a more effective strategy seems to be the prevention of biofilm formation than its eradication. The aim of the study was to examine whether the process of initial colonization of materials (glass, polyethylene terephthalate, polypropylene) by food-borne Candida sp. can be impeded by clove and thyme essential oils, used at their minimal inhibitory concentrations. In the presence of clove oil, 68.4–84.2% of the yeast tested showed a statistically significant reduction in biofilm formation, depending on the material. After treatment with thyme oil, statistically significant decrease in biofilm cell numbers was observed for 63.2–73.7% of yeasts. Confocal laser scanning microscopy showed diverse compounds of clove and thyme oils that were disparately located in C. albicans cell, on a cell wall and a cell membrane, in cytoplasm, and in vacuoles, depicting the multidirectional action of essential oils. However, essential oils that were used in sub-inhibitory concentration were sequestrated in the yeast vacuoles, which indicate the activation of Candida defense mechanisms by cell detoxification. Clove and thyme essential oils due to their anti-biofilm activity can be efficiently used in the prevention of the tested abiotic surfaces colonization by Candida sp.

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