scholarly journals Identification and Characterization of an Acinetobacter baumannii Biofilm-Associated Protein

2007 ◽  
Vol 190 (3) ◽  
pp. 1036-1044 ◽  
Author(s):  
Thomas W. Loehfelm ◽  
Nicole R. Luke ◽  
Anthony A. Campagnari
Keyword(s):  
Cell Surface ◽  
Acinetobacter Baumannii ◽  
Laser Scanning ◽  
Time Course ◽  
Surface Reactivity ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Specific Cell ◽  
Military Health ◽  
Biofilm Thickness

ABSTRACT We have identified a homologue to the staphylococcal biofilm-associated protein (Bap) in a bloodstream isolate of Acinetobacter baumannii. The fully sequenced open reading frame is 25,863 bp and encodes a protein with a predicted molecular mass of 854 kDa. Analysis of the nucleotide sequence reveals a repetitive structure consistent with bacterial cell surface adhesins. Bap-specific monoclonal antibody (MAb) 6E3 was generated to an epitope conserved among 41% of A. baumannii strains isolated during a recent outbreak in the U.S. military health care system. Flow cytometry confirms that the MAb 6E3 epitope is surface exposed. Random transposon mutagenesis was used to generate A. baumannii bap1302::EZ-Tn5, a mutant negative for surface reactivity to MAb 6E3 in which the transposon disrupts the coding sequence of bap. Time course confocal laser scanning microscopy and three-dimensional image analysis of actively growing biofilms demonstrates that this mutant is unable to sustain biofilm thickness and volume, suggesting a role for Bap in supporting the development of the mature biofilm structure. This is the first identification of a specific cell surface protein directly involved in biofilm formation by A. baumannii and suggests that Bap is involved in intercellular adhesion within the mature biofilm.

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.

The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy

Development ◽  
1992 ◽  
Vol 114 (2) ◽  
pp. 379-388 ◽  
Author(s):  
M.J. Carette ◽  
M.W. Ferguson
Keyword(s):  
Laser Scanning ◽  
Time Course ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Palatal Fusion ◽  
Morphological Criteria ◽  
Medial Edge ◽  
Mesenchymal Transformation

Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.

Tyrosine phosphorylation following alterations in arteriolar intraluminal pressure and wall tension

2001 ◽  
Vol 281 (3) ◽  
pp. H1047-H1056 ◽  
Author(s):  
Timothy V. Murphy ◽  
Brian E. Spurrell ◽  
Michael A. Hill
Keyword(s):  
Tyrosine Phosphorylation ◽  
Fluorescence Intensity ◽  
Laser Scanning ◽  
Time Course ◽  
Transmural Pressure ◽  
Laser Scanning Microscopy ◽  
Intraluminal Pressure ◽  
Confocal Laser ◽  
Wall Tension ◽  
Myogenic Activity

Arterioles respond to increased transmural pressure with myogenic constriction. The present study investigated the role of tyrosine phosphorylation in myogenic activity. Cannulated segments of a rat cremaster arteriole were fixed under pressure, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-phosphotyrosine. Smooth muscle cell fluorescence intensity was measured with the use of confocal laser-scanning microscopy. Anti-phosphotyrosine fluorescence intensity in muscle cells of arterioles maintained at 100 mmHg was reduced by the tyrosine kinase inhibitor tyrphostin A47 (30 μM) and increased by the tyrosine phosphatase inhibitor pervanadate (100 μM). In time-course experiments, anti-phosphotyrosine fluorescence increased slowly (over 5 min) after an acute increase in intraluminal pressure, and was dissociated from myogenic contraction (within 1 min). In contrast, angiotensin II (0.1 μM) caused rapid constriction and increased tyrosine phosphorylation. Anti-phosphotyrosine fluorescence was also pressure dependent (10–100 mmHg). Abolition of myogenic activity, either through removal of extracellular Ca2+, or exposure to verapamil (5 μM) or forskolin (0.1 μM) caused a further increase in anti-phosphotyrosine fluorescence. We conclude that transmural pressure and/or wall tension in arterioles causes increased tyrosine phosphorylation; however, this is not involved in the acute phase of myogenic constriction but may be involved in later responses, such as sustained myogenic tone or mechanisms possibly related to growth.

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 Effect of arsenite on swimming motility delays surface colonization in Herminiimonas arsenicoxydans

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2336-2342 ◽  
Author(s):  
M. Marchal ◽  
R. Briandet ◽  
S. Koechler ◽  
B. Kammerer ◽  
P. N. Bertin
Keyword(s):  
Laser Scanning ◽  
Time Course ◽  
Wild Type Strain ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Arsenite Oxidase ◽  
Swimming Motility ◽  
In The Wild ◽  
Biofilm Development

Herminiimonas arsenicoxydans is a Gram-negative bacterium able to detoxify arsenic-contaminated environments by oxidizing arsenite [As(III)] to arsenate [As(V)] and by scavenging arsenic ions in an extracellular matrix. Its motility and colonization behaviour have been previously suggested to be influenced by arsenite. Using time-course confocal laser scanning microscopy, we investigated its biofilm development in the absence and presence of arsenite. Arsenite was shown to delay biofilm initiation in the wild-type strain; this was partly explained by its toxicity, which caused an increased growth lag time. However, this delayed adhesion step in the presence of arsenite was not observed in either a swimming motility defective fliL mutant or an arsenite oxidase defective aoxB mutant; both strains displayed the wild-type surface properties and growth capacities. We propose that during the biofilm formation process arsenite acts on swimming motility as a result of the arsenite oxidase activity, preventing the switch between planktonic and sessile lifestyles. Our study therefore highlights the existence, under arsenite exposure, of a competition between swimming motility, resulting from arsenite oxidation, and biofilm initiation.

scholarly journals A Protein Kinase A-Dependent Mechanism by Which Rotavirus Affects the Distribution and mRNA Level of the Functional Tight Junction-Associated Protein, Occludin, in Human Differentiated Intestinal Caco-2 Cells

2007 ◽  
Vol 81 (16) ◽  
pp. 8579-8586 ◽  
Author(s):  
Isabelle Beau ◽  
Jacqueline Cotte-Laffitte ◽  
Raymonde Amsellem ◽  
Alain L. Servin
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Laser Scanning ◽  
Time Course ◽  
Mrna Level ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Infected Cells ◽  
Triton X ◽  
Kinase A

ABSTRACT We found that at the tight junctions (TJs) of Caco-2 cell monolayers, rhesus monkey rotavirus (RRV) infection induced the disappearance of occludin. Confocal laser scanning microscopy showed the disappearance of occludin from the cell-cell boundaries without modifying the expression of the other TJ-associated proteins, ZO-1 and ZO-3. Western immunoblot analysis of RRV-infected cells showed a significant fall in the levels of the nonphosphorylated form of occludin in both Triton X-100-insoluble and Triton X-100-soluble fractions, without any change in the levels of the phosphorylated form of occludin. Quantitative reverse transcription-PCRs revealed that the level of transcription of the gene that encodes occludin was significantly reduced in RRV-infected cells. Treatment of RRV-infected cells with Rp-cyclic AMP and protein kinase A inhibitors H89 and KT5720 during the time course of the infection restored the distribution of occludin and a normal level of transcription of the gene that encodes occludin.

scholarly journals Xylella fastidiosa subsp. pauca and fastidiosa Colonize Arabidopsis Systemically and Induce Anthocyanin Accumulation in Infected Leaves

2019 ◽  
Vol 109 (2) ◽  
pp. 225-232 ◽  
Author(s):  
W. E. L. Pereira ◽  
C. B. Ferreira ◽  
R. Caserta ◽  
M. Melotto ◽  
A. A. de Souza
Keyword(s):  
Laser Scanning ◽  
Time Course ◽  
Xylella Fastidiosa ◽  
Olive Tree ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Anthocyanin Accumulation ◽  
Bacterial Populations ◽  
Host Interaction ◽  
Genetic Components

The bacterium Xylella fastidiosa is a multihost pathogen that affects perennial crops such as grapevine, sweet orange, and olive tree worldwide. It is inherently difficult to study these pathosystems owing to the long-term growth habit of the host plant. Thus, the availability of model plants becomes essential to accelerate discoveries with economic impact. In this study, we uncovered evidence that the model plant Arabidopsis thaliana can be colonized by two different X. fastidiosa subspecies, pauca and fastidiosa. We observed that these bacteria are able to move away from the inoculation point as high bacterial populations were found in distant tissues. In addition, confocal laser scanning microscopy analysis of bacterial movement inside the petiole revealed the ability of the bacterium to move against the net xylem flow during the time course of colonization forming biofilm. These findings provide evidence for the capacity of X. fastidiosa to colonize Arabidopsis. Furthermore, leaves inoculated with X. fastidiosa showed a significant accumulation of anthocyanin. We propose that the X. fastidiosa subsp. pauca or fastidiosa colonization pattern and anthocyanin accumulation in the Arabidopsis ecotype Col-0 can be used as marker phenotypes to facilitate further studies aimed at improving genetic components involved in X. fastidiosa−host interaction.

scholarly journals Genetic Features of Resident Biofilms Determine Attachment of Listeria monocytogenes

2009 ◽  
Vol 75 (24) ◽  
pp. 7814-7821 ◽  
Author(s):  
Olivier Habimana ◽  
Mickael Meyrand ◽  
Thierry Meylheuc ◽  
Saulius Kulakauskas ◽  
Romain Briandet
Keyword(s):  
Listeria Monocytogenes ◽  
Laser Scanning ◽  
Time Course ◽  
Surface Hydrophobicity ◽  
Flow Cell ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Imaging ◽  
Initial Fixation ◽  
Genetic Features

ABSTRACT Planktonic Listeria monocytogenes cells in food-processing environments tend most frequently to adhere to solid surfaces. Under these conditions, they are likely to encounter resident biofilms rather than a raw solid surface. Although metabolic interactions between L. monocytogenes and resident microflora have been widely studied, little is known about the biofilm properties that influence the initial fixation of L. monocytogenes to the biofilm interface. To study these properties, we created a set of model resident Lactococcus lactis biofilms with various architectures, types of matrices, and individual cell surface properties. This was achieved using cell wall mutants that affect bacterial chain formation, exopolysaccharide (EPS) synthesis and surface hydrophobicity. The dynamics of the formation of these biofilm structures were analyzed in flow cell chambers using in situ time course confocal laser scanning microscopy imaging. All the L. lactis biofilms tested reduced the initial immobilization of L. monocytogenes compared to the glass substratum of the flow cell. Significant differences were seen in L. monocytogenes settlement as a function of the genetic background of resident lactococcal biofilm cells. In particular, biofilms of the L. lactis chain-forming mutant resulted in a marked increase in L. monocytogenes settlement, while biofilms of the EPS-secreting mutant efficiently prevented pathogen fixation. These results offer new insights into the role of resident biofilms in governing the settlement of pathogens on food chain surfaces and could be of relevance in the field of food safety controls.

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.

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