scholarly journals Biofilms' Role in Planktonic Cell Proliferation

2021 ◽  
Author(s):  
Elanna Bester ◽  
Gideon M. Wolfaardt ◽  
Nahid B. Aznaveh ◽  
Jesse Greener
Keyword(s):  
Cell Proliferation ◽  
Laser Scanning ◽  
Single Cells ◽  
Planktonic Cell ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microfluidic Channels ◽  
Shear Stresses ◽  
Biofilm Thickness ◽  
Microbial Proliferation

The detachment of single cells from biofilms is an intrinsic part of this surface-associated mode of bacterial existence. Pseudomonas sp. strain CT07gfp biofilms, cultivated in microfluidic channels under continuous flow conditions, were subjected to a range of liquid shear stresses (9.42 mPa to 320 mPa). The number of detached planktonic cells was quantified from the effluent at 24-h intervals, while average biofilm thickness and biofilm surface area were determined by confocal laser scanning microscopy and image analysis. Biofilm accumulation proceeded at the highest applied shear stress, while similar rates of planktonic cell detachment was maintained for biofilms of the same age subjected to the range of average shear rates. The conventional view of liquid-mediated shear leading to the passive erosion of single cells from the biofilm surface, disregards the active contribution of attached cell metabolism and growth to the observed detachment rates. As a complement to the conventional conceptual biofilm models, the existence of a biofilm surface-associated zone of planktonic cell proliferation is proposed to highlight the need to expand the traditional perception of biofilms as promoting microbial survival, to include the potential of biofilms to contribute to microbial proliferation.

scholarly journals Biofilms' Role in Planktonic Cell Proliferation

2021 ◽  
Author(s):  
Elanna Bester ◽  
Gideon M. Wolfaardt ◽  
Nahid B. Aznaveh ◽  
Jesse Greener
Keyword(s):  
Cell Proliferation ◽  
Laser Scanning ◽  
Single Cells ◽  
Planktonic Cell ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microfluidic Channels ◽  
Shear Stresses ◽  
Biofilm Thickness ◽  
Microbial Proliferation

The detachment of single cells from biofilms is an intrinsic part of this surface-associated mode of bacterial existence. Pseudomonas sp. strain CT07gfp biofilms, cultivated in microfluidic channels under continuous flow conditions, were subjected to a range of liquid shear stresses (9.42 mPa to 320 mPa). The number of detached planktonic cells was quantified from the effluent at 24-h intervals, while average biofilm thickness and biofilm surface area were determined by confocal laser scanning microscopy and image analysis. Biofilm accumulation proceeded at the highest applied shear stress, while similar rates of planktonic cell detachment was maintained for biofilms of the same age subjected to the range of average shear rates. The conventional view of liquid-mediated shear leading to the passive erosion of single cells from the biofilm surface, disregards the active contribution of attached cell metabolism and growth to the observed detachment rates. As a complement to the conventional conceptual biofilm models, the existence of a biofilm surface-associated zone of planktonic cell proliferation is proposed to highlight the need to expand the traditional perception of biofilms as promoting microbial survival, to include the potential of biofilms to contribute to microbial proliferation.

scholarly journals Cell Adherence and Drug Delivery from Particle Based Mesoporous Silica Films

2019 ◽  
Author(s):  
Emma Björk ◽  
Bernhard Baumann ◽  
Florian Hausladen ◽  
Rainer Wittig ◽  
mika lindén
Keyword(s):  
Drug Delivery ◽  
Laser Scanning ◽  
Single Cells ◽  
Silicon Wafers ◽  
Laser Scanning Microscopy ◽  
Drug Uptake ◽  
C2c12 Cells ◽  
Confocal Laser ◽  
Particle Sizes

Spatially and temporally controlled drug delivery is important for implant and tissue engineering applications, as the efficacy and bioavailability of the drug can be enhanced, and can also allow for drugging stem cells at different stages of development. Long-term drug delivery over weeks to months is however difficult to achieve, and coating of 3D surfaces or creating patterned surfaces is a challenge using coating techniques like spin- and dip-coating. In this study, mesoporous films consisting of SBA-15 particles grown onto silicon wafers using wet processing were evaluated as a scaffold for drug delivery. Films with various particle sizes (100 – 900 nm) and hence thicknesses were grown onto OTS-functionalized silicon wafers using a direct growth method. Precise patterning of the areas for film growth could be obtained by local removal of the OTS functionalization through laser ablation. The films were incubated with the model drug DiO, and murine myoblast cells (C2C12 cells) were seeded onto films with different particle sizes. Confocal laser scanning microscopy (CLSM) was used to study the cell growth, and a vinculin-mediated adherence of C2C12 cells on all films was verified. The successful loading of DiO into the films was confirmed by UV-vis and CLSM. It was observed that the drugs did not desorb from the particles during 24 hours in cell culture. During adherent growth on the films for 4 h, small amounts of DiO and separate particles were observed inside single cells. After 24 h, a larger number of particles and a strong DiO signal were recorded in the cells, indicating a particle mediated drug uptake. A substantial amount of DiO loaded particles were however attached on the substrate after 24 making the films attractive as a long-term reservoir for drugs on e.g. medical implants.<br>

scholarly journals Antibacterial Effects of Grape Extracts on Helicobacter pylori

2008 ◽  
Vol 75 (3) ◽  
pp. 848-852 ◽  
Author(s):  
Joseph C. Brown ◽  
Guohui Huang ◽  
Vivian Haley-Zitlin ◽  
Xiuping Jiang
Keyword(s):  
Cell Proliferation ◽  
Helicobacter Pylori ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Antibacterial Effects ◽  
Grape Skin ◽  
Agar Dilution ◽  
H Pylori ◽  
Proliferation Assays

ABSTRACT Anti-Helicobacter pylori activities were determined by agar dilution, confocal laser scanning microscopy, and cell proliferation assays following treatment with various grape extracts. Muscadine grape skin possessed the strongest activity, followed by grape synergy (skin and seed) and seed, suggesting that higher phenolic levels do not necessarily determine overall anti-H. pylori efficacy.

scholarly journals High In Vitro Antibacterial Activity of Pac-525 againstPorphyromonas gingivalisBiofilms Cultured on Titanium

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Ji-yin Li ◽  
Xue-jin Wang ◽  
Li-na Wang ◽  
Xiao-xia Ying ◽  
Xiang Ren ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Pathogenic Bacteria ◽  
Fusobacterium Nucleatum ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Average Cell ◽  
Biofilm Thickness ◽  
Live Bacteria ◽  
In Vitro Antibacterial Activity

In order to investigate the potential of short antimicrobial peptides (AMPs) as alternative antibacterial agents during the treatment of peri-implantitis, the cytotoxic activity of three short AMPs, that is, Pac-525, KSL-W, and KSL, was determined using the MTT assay. The antimicrobial activity of these AMPs, ranging in concentration from 0.0039 mg/mL to 0.5 mg/mL, against the predominant planktonic pathogens, includingStreptococcus sanguis, Fusobacterium nucleatum, andPorphyromonas gingivalis, involved in peri-implantitis was investigated. Furthermore, 2-day-oldP. gingivalisbiofilms cultured on titanium surfaces were treated with Pac-525 and subsequently observed and analysed using confocal laser scanning microscopy (CLSM). The average cell proliferation curve indicated that there was no cytotoxicity due to the three short AMPs. The minimum inhibitory concentration and minimum bactericidal concentration values of Pac-525 were 0.0625 mg/mL and 0.125 mg/mL, respectively, forP. gingivalisand 0.0078 mg/mL and 0.0156 mg/mL, respectively, forF. nucleatum. Using CLSM, we confirmed that compared to 0.1% chlorhexidine, 0.5 mg/mL of Pac-525 caused a significant decrease in biofilm thickness and a decline in the percentage of live bacteria. These data indicate that Pac-525 has unique properties that might make it suitable for the inhibition the growth of pathogenic bacteria around dental implants.

scholarly journals Localization and quantification of epidermal growth factor receptors on single cells by confocal laser scanning microscopy.

1992 ◽  
Vol 40 (9) ◽  
pp. 1353-1361 ◽  
Author(s):  
M J Good ◽  
W J Hage ◽  
C L Mummery ◽  
S W De Laat ◽  
J Boonstra
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Laser Scanning ◽  
Single Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scatchard Analysis ◽  
Epidermal Growth

We have established a method for quantifying binding of fluorescence-labeled growth factors to their receptors on single cells in situ with the confocal laser scanning microscope (CLSM). Biotinylated epidermal growth factor (EGF) coupled to phycoerythrin-labeled anti-biotin was used to compare the levels of fluorescence on three different cell types for which the number of EGF factors was known from Scatchard analysis of [125I]-EGF binding. The results showed that as few as 10,000 receptors/cell were detectable above back-ground. This method will provide a rapid and quantifiable alternative to autoradiography for ligand binding to single cells in situ.

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.

Pbx1, Meis1, and Runx1 Expression Is Decreased in the Diaphragmatic and Pulmonary Mesenchyme of Rats with Nitrofen-Induced Congenital Diaphragmatic Hernia

2020 ◽  
Author(s):  
Toshiaki Takahashi ◽  
Florian Friedmacher ◽  
Julia Zimmer ◽  
Prem Puri
Keyword(s):  
Cell Proliferation ◽  
Congenital Diaphragmatic Hernia ◽  
Diaphragmatic Hernia ◽  
Laser Scanning ◽  
Integration Site ◽  
Mesenchymal Cell ◽  
Branching Morphogenesis ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Runx1 Expression

Abstract Introduction Congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia (PH) are thought to originate from mesenchymal defects in pleuroperitoneal folds (PPFs) and primordial lungs. Pre-B-cell leukemia homeobox 1 (Pbx1), its binding partner myeloid ecotropic integration site 1 (Meis1), and runt-related transcription factor 1 (Runx1) are expressed in diaphragmatic and lung mesenchyme, functioning as transcription cofactors that modulate mesenchymal cell proliferation. Furthermore, Pbx1 −/− mice develop diaphragmatic defects and PH similar to human CDH. We hypothesized that diaphragmatic and pulmonary Pbx1, Meis1, and Runx1 expression is decreased in the nitrofen-induced CDH model. Materials and Methods Time-mated rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 48) were microdissected on D13, D15, and D18, and were divided into control and nitrofen-exposed specimens. Diaphragmatic and pulmonary gene expression levels of Pbx1, Meis1, and Runx1 were analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence-double-staining for Pbx1, Meis1, and Runx1 was combined with mesenchymal/myogenic markers Gata4 and myogenin to evaluate protein expression. Results Relative mRNA expression of Pbx1, Meis1, and Runx1 was significantly decreased in PPFs (D13), developing diaphragms/lungs (D15), and muscularized diaphragms/differentiated lungs (D18) of nitrofen-exposed fetuses compared with controls. Confocal-laser-scanning-microscopy revealed markedly diminished Pbx1, Meis1, and Runx1 immunofluorescence in diaphragmatic and pulmonary mesenchyme, associated with less proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared with controls. Conclusion Decreased Pbx1, Meis1, and Runx1 expression during diaphragmatic development and lung branching morphogenesis may reduce mesenchymal cell proliferation, causing malformed PPFs and disrupted airway branching, thus leading to diaphragmatic defects and PH in the nitrofen-induced CDH model.

Confocal Laser Microscopy of Physiologically Characterized Fluorescently Labeled Central Nervous System Neurons

1988 ◽  
Vol 46 ◽  
pp. 274-275
Author(s):  
J.N. Turner ◽  
J. Swann ◽  
K. Smith ◽  
M. Siemens ◽  
D. Szarowski ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Laser Scanning ◽  
Tissue Sample ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Brain Slices ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Efficient Detection

Confocal laser scanning microscopy (CLSM) is capable of three-dimensional imaging of fluorescently labeled single cells. Efficient detection via a photomultiplier and optical sectioning with high rejection of light from other specimen levels make it possible to image cells surrounded by either labeled or unlabeled tissue. It is no longer necessary to restrict high resolution light microscopy to cultured cells or those near the surface of a tissue sample. Cells can be observed üin situ” in a physiologically characterized environment. Central nervous system neurons can be electrophysiologically characterized and then injected with a fluorescent dye such as lucifer yellow. The CLSM can excite the dye and image the fluorescent emission in thick tissue preparations (hundreds of micrometers) making possible a new approach to the correlation of physiology and anatomy.Brain slices 350 μm thick were obtained from hippocampus and inferior colliculus of immature rats and incubated in oxygenated artificial cerebrospinal fluid. Cells were penetrated with micropipets, characterized electrophysiologically and ionophoretically injected with 5% lucifer yellow in LiAc.

scholarly journals Microscopic and Spectroscopic Analyses of Chlorhexidine Tolerance in Delftia acidovorans Biofilms

2014 ◽  
Vol 58 (10) ◽  
pp. 5673-5686 ◽  
Author(s):  
Tara Rema ◽  
John R. Lawrence ◽  
James J. Dynes ◽  
Adam P. Hitchcock ◽  
Darren R. Korber
Keyword(s):  
Laser Scanning ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Content Type ◽  
Flow Cells ◽  
Biofilm Thickness ◽  
Delftia Acidovorans ◽  
Scanning Transmission ◽  
Negative Effect

ABSTRACTThe physicochemical responses ofDelftia acidovoransbiofilms exposed to the commonly used antimicrobial chlorhexidine (CHX) were examined in this study. A CHX-sensitive mutant (MIC, 1.0 μg ml−1) was derived from a CHX-tolerant (MIC, 15.0 μg ml−1)D. acidovoransparent strain using transposon mutagenesis.D. acidovoransmutant (MT51) and wild-type (WT15) strain biofilms were cultivated in flow cells and then treated with CHX at sub-MIC and inhibitory concentrations and examined by confocal laser scanning microscopy (CLSM), scanning transmission X-ray microscopy (STXM), and infrared (IR) spectroscopy. Specific morphological, structural, and chemical compositional differences between the CHX-treated and -untreated biofilms of both strains were observed. Apart from architectural differences, CLSM revealed a negative effect of CHX on biofilm thickness in the CHX-sensitive MT51 biofilms relative to those of the WT15 strain. STXM analyses showed that the WT15 biofilms contained two morphochemical cell variants, whereas only one type was detected in the MT51 biofilms. The cells in the MT51 biofilms bioaccumulated CHX to a similar extent as one of the cell types found in the WT15 biofilms, whereas the other cell type in the WT15 biofilms did not bioaccumulate CHX. STXM and IR spectral analyses revealed that CHX-sensitive MT51 cells accumulated the highest levels of CHX. Pretreating biofilms with EDTA promoted the accumulation of CHX in all cells. Thus, it is suggested that a subpopulation of cells that do not accumulate CHX appear to be responsible for greater CHX resistance inD. acidovoransWT15 biofilm in conjunction with the possible involvement of bacterial membrane stability.

scholarly journals Optimizing CNT Loading in Antimicrobial Composites for Urinary Tract Application

2021 ◽  
Vol 11 (9) ◽  
pp. 4038
Author(s):  
Marisa Gomes ◽  
Luciana C. Gomes ◽  
Rita Teixeira-Santos ◽  
Manuel F. R. Pereira ◽  
Olívia S. G. P. Soares ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Laser Scanning ◽  
Antimicrobial Properties ◽  
Surface Hydrophobicity ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Coating Materials ◽  
Biofilm Thickness ◽  
Angle Measurements ◽  
Antifouling Coatings

Several methodologies have been implemented with the intent of preventing or reducing the formation of biofilms on indwelling urinary devices. The use of carbon nanotubes (CNTs) in the biomedical field has been increasing, particularly in the production of antimicrobial and antifouling coatings. Despite their proven antimicrobial properties, their use as coating materials for urinary tract devices (UTDs) is still poorly documented. In the present work, CNT/poly(dimethylsiloxane) (PDMS) composite materials containing different CNT loadings were prepared and further tested against Escherichia coli under conditions prevailing in UTDs. Besides CNT loading optimization, textural modifications were also introduced on the surface of CNTs to improve the antibiofilm pro-perties of the final composites. Material characterization included the textural evaluation of CNTs and the assessment of surface morphology by scanning electron microscopy, while the surface hydrophobicity was determined by contact angle measurements. Biofilm analysis was performed by determining the number of culturable and total cells and by confocal laser scanning microscopy. Results revealed that, by filling the PDMS matrix with 3 wt% CNT loading, a significant reduction in cell culturability (39%) can be achieved compared to PDMS. Additionally, the textural modifications induced by ball-milling treatment proved to be effective on the inhibition of biofilm formation, reducing the amount of biofilm per surface area, biofilm thickness and surface coverage in 31, 47 and 27%, respectively (compared to surfaces where CNTs were not ball-milled).

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