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 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 Gentian Violet Exhibits Activity against Biofilms Formed by Oral Candida Isolates Obtained from HIV-Infected Patients

2011 ◽  
Vol 55 (6) ◽  
pp. 3043-3045 ◽  
Author(s):  
Rana S. Traboulsi ◽  
Pranab K. Mukherjee ◽  
Jyotsna Chandra ◽  
Robert A. Salata ◽  
Richard Jurevic ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Laser Scanning ◽  
Gentian Violet ◽  
Dry Weight ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Content Type ◽  
Biofilm Thickness

ABSTRACTThe effect of gentian violet againstCandida albicansand non-Candida albicansbiofilms formed on polymethylmethacrylate strips was evaluated using a dry weight assay and confocal laser scanning microscopy. The ability of gentian violet to inhibitCandida albicansgermination was also assessed. Gentian violet activity againstCandidabiofilms was demonstrated by a reduction in dry weight, disruption of biofilm architecture, and reduced biofilm thickness. Additionally, gentian violet inhibitedCandidagermination in a concentration-dependent manner.

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 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.

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 SipA Activation of Caspase-3 Is a Decisive Mediator of Host Cell Survival at Early Stages of Salmonella enterica Serovar Typhimurium Infection

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Anne McIntosh ◽  
Lynsey M. Meikle ◽  
Michael J. Ormsby ◽  
Beth A. McCormick ◽  
John M. Christie ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Actin Polymerization ◽  
Caspase 3 ◽  
Ex Vivo ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Limiting Factor ◽  
Dual Function ◽  
Content Type

ABSTRACT Salmonella invasion protein A (SipA) is a dual-function effector protein that plays roles in both actin polymerization and caspase-3 activation in intestinal epithelial cells. To date its function in other cell types has remained largely unknown despite its expression in multiple cell types and its extracellular secretion during infection. Here we show that in macrophages SipA induces increased caspase-3 activation early in infection. This activation required a threshold level of SipA linked to multiplicity of infection and may be a limiting factor controlling bacterial numbers in infected macrophages. In polymorphonuclear leukocytes, SipA or other Salmonella pathogenicity island 1 effectors had no effect on induction of caspase-3 activation either alone or in the presence of whole bacteria. Tagging of SipA with the small fluorescent phiLOV tag, which can pass through the type three secretion system, allowed visualization and quantification of caspase-3 activation by SipA-phiLOV in macrophages. Additionally, SipA-phiLOV activation of caspase-3 could be tracked in the intestine through multiphoton laser scanning microscopy in an ex vivo intestinal model. This allowed visualization of areas where the intestinal epithelium had been compromised and demonstrated the potential use of this fluorescent tag for in vivo tracking of individual effectors.

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.

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.

scholarly journals Cyanobacterial calcification in modern microbialites at the submicrometer-scale

2013 ◽  
Vol 10 (2) ◽  
pp. 3311-3339 ◽  
Author(s):  
E. Couradeau ◽  
K. Benzerara ◽  
E. Gérard ◽  
I. Estève ◽  
D. Moreira ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Morphology ◽  
Laser Scanning ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Advanced Stages

Abstract. The search for microfossils in the geological record has been a long-term challenge. Part of the problem comes from the difficulty of identifying such microfossils unambiguously, since they can be morphologically confused with abiotic biomorphs. One route to improve our ability to correctly identify microfossils consists in studying fossilization processes affecting bacteria in modern settings. We studied the initial stages of fossilization of cyanobacterial cells in modern microbialites from Lake Alchichica (Mexico), a Mg-rich hyperalkaline crater lake (pH 8.9) hosting currently growing stromatolites composed of aragonite [CaCO3] and hydromagnesite [Mg5(CO3)4(OH)2 × 4(H2O)]. Most of the biomass associated with the microbialites is composed of cyanobacteria. Scanning electron microscopy analyses coupled with confocal laser scanning microscopy observations were conducted to co-localize cyanobacterial cells and associated minerals. These observations showed that cyanobacterial cells affiliating to the order Pleurocapsales become specifically encrusted within aragonite with an apparent preservation of cell morphology. Encrustation gradients from non-encrusted to totally encrusted cells spanning distances of a few hundred micrometers were observed. Cells exhibiting increased levels of encrustation along this gradient were studied down to the nm-scale using a combination of focused ion beam (FIB) milling, transmission electron microscopy (TEM) and scanning transmission X-ray microscopy (STXM) at the C, O and N K-edges. Two different types of aragonite crystals were observed: one type was composed of needle-shaped nano-crystals growing outward from the cell body with a crystallographic orientation perpendicular to the cell wall, and another type was composed of larger crystals that progressively filled the cell interior. Organic matter (OM), initially co-localized with the cell, decreased in concentration and dispersed away from the cell while crystal growth occurred. As encrustation developed, OM progressively disappeared, but remaining OM showed the same spectroscopic signature. In the most advanced stages of fossilization, only the textural organization of the two types of aragonite recorded the initial cell morphology and spatial distribution.

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