scholarly journals Hydrosol of Thymbra capitata Is a Highly Efficient Biocide against Salmonella enterica Serovar Typhimurium Biofilms

2016 ◽  
Vol 82 (17) ◽  
pp. 5309-5319 ◽  
Author(s):  
Foteini Karampoula ◽  
Efstathios Giaouris ◽  
Julien Deschamps ◽  
Agapi I. Doulgeraki ◽  
George-John E. Nychas ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Laser Scanning ◽  
High Efficiency ◽  
3D Structure ◽  
Time Lapse ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Planktonic Cells ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTSalmonellais recognized as one of the most significant enteric foodborne bacterial pathogens. In recent years, the resistance of pathogens to biocides and other environmental stresses, especially when they are embedded in biofilm structures, has led to the search for and development of novel antimicrobial strategies capable of displaying both high efficiency and safety. In this direction, the aims of the present work were to evaluate the antimicrobial activity of hydrosol of the Mediterranean spiceThymbracapitataagainst both planktonic and biofilm cells ofSalmonella entericaserovar Typhimurium and to compare its action with that of benzalkonium chloride (BC), a commonly used industrial biocide. In order to achieve this, the disinfectant activity following 6-min treatments was comparatively evaluated for both disinfectants by calculating the concentrations needed to achieve the same log reductions against both types of cells. Their bactericidal effect against biofilm cells was also comparatively determined byin situand real-time visualization of cell inactivation through the use of time-lapse confocal laser scanning microscopy (CLSM). Interestingly, results revealed that hydrosol was almost equally effective against biofilms and planktonic cells, whereas a 200-times-higher concentration of BC was needed to achieve the same effect against biofilm compared to planktonic cells. Similarly, time-lapse CLSM revealed the significant advantage of the hydrosol to easily penetrate within the biofilm structure and quickly kill the cells, despite the three-dimensional (3D) structure ofSalmonellabiofilm.IMPORTANCEThe results of this paper highlight the significant antimicrobial action of a natural compound, hydrosol ofThymbra capitata, against both planktonic and biofilm cells of a common foodborne pathogen. Hydrosol has numerous advantages as a disinfectant of food-contact surfaces. It is an aqueous solution which can easily be rinsed out from surfaces, it does not have the strong smell of the essential oil (EO) and it is a byproduct of the EO distillation procedure without any industrial application until now. Consequently, hydrosol obviously could be of great value to combat biofilms and thus to improve product safety not only for the food industries but probably also for many other industries which experience biofilm-related problems.

scholarly journals The Protective Effect of Staphylococcus epidermidis Biofilm Matrix against Phage Predation

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1076
Author(s):  
Luís D. R. Melo ◽  
Graça Pinto ◽  
Fernando Oliveira ◽  
Diana Vilas-Boas ◽  
Carina Almeida ◽  
...  
Keyword(s):  
Protective Effect ◽  
Staphylococcus Epidermidis ◽  
Laser Scanning ◽  
3D Structure ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Planktonic Cells ◽  
The Matrix ◽  
The Impact ◽  
Biofilm Matrix

Staphylococcus epidermidis is a major causative agent of nosocomial infections, mainly associated with the use of indwelling devices, on which this bacterium forms structures known as biofilms. Due to biofilms’ high tolerance to antibiotics, virulent bacteriophages were previously tested as novel therapeutic agents. However, several staphylococcal bacteriophages were shown to be inefficient against biofilms. In this study, the previously characterized S. epidermidis-specific Sepunavirus phiIBB-SEP1 (SEP1), which has a broad spectrum and high activity against planktonic cells, was evaluated concerning its efficacy against S. epidermidis biofilms. The in vitro biofilm killing assays demonstrated a reduced activity of the phage. To understand the underlying factors impairing SEP1 inefficacy against biofilms, this phage was tested against distinct planktonic and biofilm-derived bacterial populations. Interestingly, SEP1 was able to lyse planktonic cells in different physiological states, suggesting that the inefficacy for biofilm control resulted from the biofilm 3D structure and the protective effect of the matrix. To assess the impact of the biofilm architecture on phage predation, SEP1 was tested in disrupted biofilms resulting in a 2 orders-of-magnitude reduction in the number of viable cells after 6 h of infection. The interaction between SEP1 and the biofilm matrix was further assessed by the addition of matrix to phage particles. Results showed that the matrix did not inactivate phages nor affected phage adsorption. Moreover, confocal laser scanning microscopy data demonstrated that phage infected cells were less predominant in the biofilm regions where the matrix was more abundant. Our results provide compelling evidence indicating that the biofilm matrix can work as a barrier, allowing the bacteria to be hindered from phage infection.

scholarly journals Calcite Biomineralization by Bacterial Isolates from the Recently Discovered Pristine Karstic Herrenberg Cave

2011 ◽  
Vol 78 (4) ◽  
pp. 1157-1167 ◽  
Author(s):  
Anna Rusznyák ◽  
Denise M. Akob ◽  
Sándor Nietzsche ◽  
Karin Eusterhues ◽  
Kai Uwe Totsche ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Extracellular Polymeric Substances ◽  
Large Fraction ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Rrna Gene ◽  
Seepage Water ◽  
Bacterial Cells ◽  
Content Type ◽  
Rich Media

ABSTRACTKarstic caves represent one of the most important subterranean carbon storages on Earth and provide windows into the subsurface. The recent discovery of the Herrenberg Cave, Germany, gave us the opportunity to investigate the diversity and potential role of bacteria in carbonate mineral formation. Calcite was the only mineral observed by Raman spectroscopy to precipitate as stalactites from seepage water. Bacterial cells were found on the surface and interior of stalactites by confocal laser scanning microscopy. Proteobacteria dominated the microbial communities inhabiting stalactites, representing more than 70% of total 16S rRNA gene clones. Proteobacteria formed 22 to 34% of the detected communities in fluvial sediments, and a large fraction of these bacteria were also metabolically active. A total of 9 isolates, belonging to the generaArthrobacter,Flavobacterium,Pseudomonas,Rhodococcus,Serratia, andStenotrophomonas, grew on alkaline carbonate-precipitating medium. Two cultures with the most intense precipitate formation,Arthrobacter sulfonivoransandRhodococcus globerulus, grew as aggregates, produced extracellular polymeric substances (EPS), and formed mixtures of calcite, vaterite, and monohydrocalcite.R. globerulusformed idiomorphous crystals with rhombohedral morphology, whereasA. sulfonivoransformed xenomorphous globular crystals, evidence for taxon-specific crystal morphologies. The results of this study highlighted the importance of combining various techniques in order to understand the geomicrobiology of karstic caves, but further studies are needed to determine whether the mineralogical biosignatures found in nutrient-rich media can also be found in oligotrophic caves.

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 Visualization of the Peroxisomal Compartment in Living Mammalian Cells: Dynamic Behavior and Association with Microtubules

1997 ◽  
Vol 136 (1) ◽  
pp. 71-80 ◽  
Author(s):  
Erik A.C. Wiemer ◽  
Thibaut Wenzel ◽  
Thomas J. Deerinck ◽  
Mark H. Ellisman ◽  
Suresh Subramani
Keyword(s):  
Mammalian Cells ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Small Subset ◽  
Subcellular Compartment ◽  
Directional Movement ◽  
Green Fluorescent

Peroxisomes in living CV1 cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP–PTS1). The organelle dynamics were examined and analyzed using time-lapse confocal laser scanning microscopy. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (∼95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (∼5%) of the peroxisomes. In the latter instance, peak velocities up to 0.75 μm/s and sustained directional velocities up to 0.45 μm/s over 11.5 μm were recorded. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome morphology and subcellular distribution and blocked the directional movement. In contrast, the microtubule-stabilizing compound paclitaxel, or the microfilament-destabilizing drugs cytochalasin B or D, did not exert these effects. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis.

scholarly journals Alginate Oligosaccharide-Induced Modification of the lasI-lasR and rhlI-rhlR Quorum-Sensing Systems in Pseudomonas aeruginosa

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Alison A. Jack ◽  
Saira Khan ◽  
Lydia C. Powell ◽  
Manon F. Pritchard ◽  
Konrad Beck ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Laser Scanning ◽  
Homoserine Lactone ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Mass Spectrometry Analysis ◽  
Confocal Laser ◽  
Steric Interaction ◽  
Content Type

ABSTRACT Pseudomonas aeruginosa plays a major role in many chronic infections. Its ability to readily form biofilms contributes to its success as an opportunistic pathogen and its resistance/tolerance to antimicrobial/antibiotic therapy. A low-molecular-weight alginate oligomer (OligoG CF-5/20) derived from marine algae has previously been shown to impair motility in P. aeruginosa biofilms and disrupt pseudomonal biofilm assembly. As these bacterial phenotypes are regulated by quorum sensing (QS), we hypothesized that OligoG CF-5/20 may induce alterations in QS signaling in P. aeruginosa . QS regulation was studied by using Chromobacterium violaceum CV026 biosensor assays that showed a significant reduction in acyl homoserine lactone (AHL) production following OligoG CF-5/20 treatment (≥2%; P < 0.05). This effect was confirmed by liquid chromatography-mass spectrometry analysis of C 4 -AHL and 3-oxo-C 12 -AHL production (≥2%; P < 0.05). Moreover, quantitative PCR showed that reduced expression of both the las and rhl systems was induced following 24 h of treatment with OligoG CF-5/20 (≥0.2%; P < 0.05). Circular dichroism spectroscopy indicated that these alterations were not due to steric interaction between the AHL and OligoG CF-5/20. Confocal laser scanning microscopy (CLSM) and COMSTAT image analysis demonstrated that OligoG CF-5/20-treated biofilms had a dose-dependent decrease in biomass that was associated with inhibition of extracellular DNA synthesis (≥0.5%; P < 0.05). These changes correlated with alterations in the extracellular production of the pseudomonal virulence factors pyocyanin, rhamnolipids, elastase, and total protease ( P < 0.05). The ability of OligoG CF-5/20 to modify QS signaling in P. aeruginosa PAO1 may influence critical downstream functions such as virulence factor production and biofilm formation.

Migration of Salmonella Enteritidis Phage Type 30 through Almond Hulls and Shells

2008 ◽  
Vol 71 (2) ◽  
pp. 397-401 ◽  
Author(s):  
MICHELLE D. DANYLUK ◽  
MARIA T. BRANDL ◽  
LINDA J. HARRIS
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Salmonella Enteritidis ◽  
Fluorescent Protein ◽  
Phage Type ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Aqueous Environment ◽  
Serovar Typhimurium ◽  
Green Fluorescent

The ability of Salmonella to migrate from an external aqueous environment through the almond hull and shell, and to colonize the kernel, was evaluated in two ways. First, the outer surface of shell halves from five varieties of almonds that differed in shell hardness were placed in contact with a suspension of Salmonella enterica serovar Enteritidis phage type 30 for 24hat24°C. Salmonella Enteritidis was isolated from the inside of these almond shells in 46 and 100% of the samples, by direct swabbing of the inner surface of the shell and by enrichment from the swab, respectively. These findings suggested that hardness of the shell is not a significant factor in the migration of the pathogen through that tissue. In addition, both motile and nonmotile strains of S. enterica serovar Typhimurium migrated through the almond shells to the same extent under the conditions of this assay, indicating that bacterial migration through the wet shell may be a passive process. Second, whole almonds (intact hull, shell, and kernel) were soaked for 24 to 72 h at 24°C in a suspension of Salmonella Enteritidis phage type 30 labeled with the green fluorescent protein. Green fluorescent protein–labeled Salmonella cells were observed on the outer and inner surfaces of both the almond hull and shell, and on the kernel, by confocal laser scanning microscopy. Our data provide direct evidence that wet conditions allow for Salmonella migration through the hull and shell and onto the almond kernel, thus providing a means by which almond kernels may become contaminated in the field.

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 Dynamics of the Action of Biocides in Pseudomonas aeruginosa Biofilms

2011 ◽  
Vol 55 (6) ◽  
pp. 2648-2654 ◽  
Author(s):  
A. Bridier ◽  
F. Dubois-Brissonnet ◽  
G. Greub ◽  
V. Thomas ◽  
R. Briandet
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Biochemical Analysis ◽  
Laser Scanning Microscopy ◽  
Diffusion Reaction ◽  
Confocal Laser ◽  
Staining Procedure ◽  
Fluorescence Staining ◽  
Content Type ◽  
Real Time Visualization

ABSTRACTThe biocidal activity of peracetic acid (PAA) and benzalkonium chloride (BAC) onPseudomonas aeruginosabiofilms was investigated by using a recently developed confocal laser scanning microscopy (CLSM) method that enables the direct and real-time visualization of cell inactivation within the structure. This technique is based on monitoring the loss of fluorescence that corresponds to the leakage of a fluorophore out of cells due to membrane permeabilization by the biocides. Although this approach has previously been used with success with various Gram-positive species, it is not directly applicable to the visualization of Gram-negative strains such asP. aeruginosa, particularly because of limitations regarding fluorescence staining. After adapting the staining procedure toP. aeruginosa, the action of PAA and BAC on the biofilm formed by strain ATCC 15442 was investigated. The results revealed specific inactivation patterns as a function of the mode of action of the biocides. While PAA treatment triggered a uniform loss of fluorescence in the structure, the action of BAC was first localized at the periphery of cell clusters and then gradually spread throughout the biofilm. Visualization of the action of BAC in biofilms formed by three clinical isolates then confirmed the presence of a delay in penetration, showing that diffusion-reaction limitations could provide a major explanation for the resistance ofP. aeruginosabiofilms to this biocide. Biochemical analysis suggested a key role for extracellular matrix characteristics in these processes.

Toxicity Evaluation of Two Dental Composites: Three-Dimensional Confocal Laser Scanning Microscopy Time-Lapse Imaging of Cell Behavior

2013 ◽  
Vol 19 (3) ◽  
pp. 596-607 ◽  
Author(s):  
Ghania Nina Attik ◽  
Nelly Pradelle-Plasse ◽  
Doris Campos ◽  
Pierre Colon ◽  
Brigitte Grosgogeat
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Cell Metabolism ◽  
Three Dimensional ◽  
Time Lapse ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Dental Composites ◽  
Confocal Laser ◽  
Time Lapse Imaging

AbstractThe purpose of this study was to investigate thein vitrobiocompatibility of two dental composites (namely A and B) with similar chemical composition used for direct restoration using three-dimensional confocal laser scanning microscopy (CLSM) time-lapse imaging. Time-lapse imaging was performed on cultured human HGF-1 fibroblast-like cells after staining using Live/Dead®. Image analysis showed a higher mortality rate in the presence of composite A than composite B. The viability rate decreased in a time-dependent manner during the 5 h of exposure. Morphological alterations were associated with toxic effects; cells were enlarged and more rounded in the presence of composite A as shown by F-actin and cell nuclei staining. Resazurin assay was used to confirm the active potential of composites in cell metabolism; results showed severe cytotoxic effects in the presence of both no light-curing composites after 24 h of direct contact. However, extracts of polymerized composites induced a moderate decrease in cell metabolism after the same incubation period. Composite B was significantly better tolerated than composite A at all investigated end points and all time points. The finding confirmed that the used CLSM method was sufficiently sensitive to differentiate the biocompatibility behavior of two composites based on similar methacrylate monomers.

scholarly journals Enhancement of Vancomycin Activity against Biofilms by Using Ultrasound-Targeted Microbubble Destruction

2011 ◽  
Vol 55 (11) ◽  
pp. 5331-5337 ◽  
Author(s):  
Nianan He ◽  
Jian Hu ◽  
Huayong Liu ◽  
Tao Zhu ◽  
Beijian Huang ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Laser Scanning ◽  
Rabbit Model ◽  
Antibiotic Activity ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Content Type ◽  
Implanted Medical Devices

ABSTRACTTreating biofilm infections on implanted medical devices is formidable, even with extensive antibiotic therapy. The aim of this study was to investigate whether ultrasound (US)-targeted microbubble (MB) destruction (UTMD) could enhance vancomycin activity againstStaphylococcus epidermidisRP62A biofilms. Twelve-hour biofilms were treated with vancomycin combined with UTMD. The vancomycin and MB (SonoVue) were used at concentrations of 100 μg/ml and 30% (vol/vol), respectively, in studiesin vitro. After US exposure (0.08 MHz, 1.0 W/cm2, 50% duty cycle, and 10-min duration), the biofilms were cultured at 37°C for another 12 h. The results showed that many micropores were found in biofilms treated with vancomycin combined with UTMD. Biofilm densities (A570values) and the viable counts ofS. epidermidisrecovered from the biofilm were significantly decreased compared with those of any other groups. Furthermore, the highest percentage of dead cells was found, using confocal laser scanning microscopy, in the biofilm treated with vancomycin combined with UTMD. The viable counts of bacteria in biofilms in anin vivorabbit model also confirmed the enhanced effect of vancomycin combined with UTMD. UTMD may have great potential for improving antibiotic activity against biofilm infections.

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