scholarly journals N-Acetyl-l-Cysteine and Cysteamine as New Strategies against Mixed Biofilms of Nonencapsulated Streptococcus pneumoniae and Nontypeable Haemophilus influenzae

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Mirian Domenech ◽  
Ernesto García
Keyword(s):  
Streptococcus Pneumoniae ◽  
Haemophilus Influenzae ◽  
Laser Scanning ◽  
Acute Otitis Media ◽  
Public Health Problem ◽  
Fluorescent Labeling ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nontypeable Haemophilus Influenzae ◽  
Content Type

ABSTRACT Acute otitis media, a polymicrobial disease of the middle ear cavity of children, is a significant public health problem worldwide. It is most frequently caused by encapsulated Streptococcus pneumoniae and nontypeable Haemophilus influenzae, although the widespread use of pneumococcal conjugate vaccines is apparently producing an increase in the carriage of nonencapsulated S. pneumoniae. Frequently, pneumococci and H. influenzae live together in the human nasopharynx, forming a self-produced biofilm. Biofilms present a global medical challenge since the inherent antibiotic resistance of their producers demands the use of large doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence. Here, we describe the development of an in vitro nonencapsulated S. pneumoniae-nontypeable H. influenzae biofilm system with polystyrene or glass-bottom plates. Confocal laser scanning microscopy and specific fluorescent labeling of pneumococcal cells with Helix pomatia agglutinin revealed an even distribution of both species within the biofilm. This simple and robust protocol of mixed biofilms was used to test the antimicrobial properties of two well-known antioxidants that are widely used in the clinical setting, i.e., N-acetyl-l-cysteine and cysteamine. This repurposing approach showed the high potency of N-acetyl-l-cysteine and cysteamine against mixed biofilms of nonencapsulated S. pneumoniae and nontypeable H. influenzae. Decades of clinical use mean that these compounds are safe to use, which may accelerate their evaluation in humans.

scholarly journals Following Pathogen Development and Gene Expression in a Food Ecosystem: the Case of a Staphylococcus aureus Isolate in Cheese

2014 ◽  
Vol 80 (16) ◽  
pp. 5106-5115 ◽  
Author(s):  
Isabelle Fleurot ◽  
Marina Aigle ◽  
Renaud Fleurot ◽  
Claire Darrigo ◽  
Jacques-Antoine Hennekinne ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Laser Scanning ◽  
Public Health Problem ◽  
Laser Scanning Microscopy ◽  
Solid Matrix ◽  
Confocal Laser ◽  
Natural Food ◽  
Content Type ◽  
Food Isolate

ABSTRACTHuman intoxication or infection due to bacterial food contamination constitutes an economic challenge and a public health problem. Information on thein situdistribution and expression of pathogens responsible for this risk is to date lacking, largely because of technical bottlenecks in detecting signals from minority bacterial populations within a complex microbial and physicochemical ecosystem. We simulated the contamination of a real high-risk cheese with a natural food isolate ofStaphylococcus aureus, an enterotoxin-producing pathogen responsible for food poisoning. To overcome the problem of a detection limit in a solid matrix, we chose to work with a fluorescent reporter (superfolder green fluorescent protein) that would allow spatiotemporal monitoring ofS. aureuspopulations and targeted gene expression. The combination of complementary techniques revealed thatS. aureuslocalizes preferentially on the cheese surface during ripening. Immunochemistry and confocal laser scanning microscopy enabled us to visualize, in a single image, dairy bacteria and pathogen populations, virulence gene expression, and the toxin produced. This procedure is readily applicable to other genes of interest, other bacteria, and different types of food matrices.

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 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.

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 Food Microstructure and Fat Content Affect Growth Morphology, Growth Kinetics, and Preferred Phase for Cell Growth ofListeria monocytogenesin Fish-Based Model Systems

2019 ◽  
Vol 85 (16) ◽  
Author(s):  
Davy Verheyen ◽  
Xiang Ming Xu ◽  
Marlies Govaert ◽  
Maria Baka ◽  
Torstein Skåra ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Microbial Growth ◽  
Growth Dynamics ◽  
Growth Behavior ◽  
Laser Scanning Microscopy ◽  
Model Systems ◽  
Confocal Laser ◽  
Growth Morphology ◽  
Content Type ◽  
Food Model

ABSTRACTFood microstructure significantly affects microbial growth dynamics, but knowledge concerning the exact influencing mechanisms at a microscopic scale is limited. The food microstructural influence onListeria monocytogenes(green fluorescent protein strain) growth at 10°C in fish-based food model systems was investigated by confocal laser scanning microscopy. The model systems had different microstructures, i.e., liquid, xanthan (high-viscosity liquid), aqueous gel, and emulsion and gelled emulsion systems varying in fat content. Bacteria grew as single cells, small aggregates, and microcolonies of different sizes (based on colony radii [size I, 1.5 to 5.0 μm; size II, 5.0 to 10.0 μm; size III, 10.0 to 15.0 μm; and size IV, ≥15 μm]). In the liquid, small aggregates and size I microcolonies were predominantly present, while size II and III microcolonies were predominant in the xanthan and aqueous gel. Cells in the emulsions and gelled emulsions grew in the aqueous phase and on the fat-water interface. A microbial adhesion to solvent assay demonstrated limited bacterial nonpolar solvent affinities, implying that this behavior was probably not caused by cell surface hydrophobicity. In systems containing 1 and 5% fat, the largest cell volume was mainly represented by size I and II microcolonies, while at 10 and 20% fat a few size IV microcolonies comprised nearly the total cell volume. Microscopic results (concerning, e.g., growth morphology, microcolony size, intercolony distances, and the preferred phase for growth) were related to previously obtained macroscopic growth dynamics in the model systems for anL. monocytogenesstrain cocktail, leading to more substantiated explanations for the influence of food microstructural aspects on lag phase duration and growth rate.IMPORTANCEListeria monocytogenesis one of the most hazardous foodborne pathogens due to the high fatality rate of the disease (i.e., listeriosis). In this study, the growth behavior ofL. monocytogeneswas investigated at a microscopic scale in food model systems that mimic processed fish products (e.g., fish paté and fish soup), and the results were related to macroscopic growth parameters. Many studies have previously focused on the food microstructural influence on microbial growth. The novelty of this work lies in (i) the microscopic investigation of products with a complex composition and/or structure using confocal laser scanning microscopy and (ii) the direct link to the macroscopic level. Growth behavior (i.e., concerning bacterial growth morphology and preferred phase for growth) was more complex than assumed in common macroscopic studies. Consequently, the effectiveness of industrial antimicrobial food preservation technologies (e.g., thermal processing) might be overestimated for certain products, which may have critical food safety implications.

scholarly journals 1640. Toward New Anti-Biofilm Therapies: High Mobility Group Box 1 (HMGB1) Protein and Its Structural Variants Can Be Used to Disrupt Bacterial Biofilms (BBs)

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S46-S46
Author(s):  
Aspasia Katragkou ◽  
Lauren Warren ◽  
John Buzzo ◽  
Steven Goodman
Keyword(s):  
Laser Scanning ◽  
Structural Integrity ◽  
Public Health Problem ◽  
Full Length ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Major Public Health Problem ◽  
Average Thickness ◽  
Research Support

Abstract Background BB-related infections are a major public health problem, as they are notoriously refractory to current treatments. One of the defining characteristics of BBs is the extracellular polymeric substance (EPS). Extracellular DNA and the bacterial DNABII family of proteins are key components of EPS and are crucial for BBs structural integrity. It is known that targeting DNABII proteins disrupts BBs. We hypothesized that HMGB1, a DNA-binding eukaryotic protein, could affect BBs as it binds to the same DNA structures as the DNABII proteins. HMGB1 is comprised of 3 domains, A Box, B Box, and C tail, all of which have different functions. We aimed to determine in vitro the effects of HMGB1 and its individual domains against BBs. Methods Klebsiella pneumoniae (KP), a common cause of nosocomial infections, was used for all BBs disruption assays. Human recombinant full-length HMGB1 (rHMGB1; 1–215), a C45S mutation variant (mHMGB1) and the HMGB1 domains A Box (1–89), B Box (90–176), AB Boxes (1–176), B-linker Box (80–179), and B-linker Box C106S were expressed (in E. coli) and purified to &gt;95%. To evaluate the effect of rHMGB1 and the various domains on established BBs, each protein species (200 nM) was added to preformed BBs at 24 hours. At 40 hours the BBs were washed, stained with LIVE/DEAD®, visualized via confocal laser scanning microscopy and images were analyzed by COMSTAT to calculate average thickness and biomass. Results Exogenous rHMGB1 and its individual domains, with the exception of A Box caused a significant reduction (P &lt; 0.05) in average thickness (AT) and biomass (BM) of KP biofilms when compared with untreated KP biofilms (% reduction mean ± SE in AT: 44% ± 0.33, 75% ± 0.04, 63% ± 0.1, 77% ± 0.03, 64% ± 0.08, 54% ± 0.15 and in BM: 61% ± 0.01, 80% ± 0.01, 68% ± 0.02, 67% ± 0.01, 73% ± 0.02, 56% ± 0.02 induced by rHMGB1, mHMGB1, B-Box, B-linker Box, AB Boxes, and B-linker Box C106S, respectively). Conclusion Full-length recombinant HMGB1 was able to significantly disrupt established KP biofilms as were all truncated HMGB1 forms containing the B Box domain and could potentially be used as a therapeutic treatment for BB-related infections. Disclosures J. Buzzo, ProclaRx: Collaborator, Research support. S. Goodman, ProclaRx: Collaborator and Scientific Advisor, Research support.

scholarly journals Possible Overestimation of Surface Disinfection Efficiency by Assessment Methods Based on Liquid Sampling Procedures as Demonstrated byIn SituQuantification of Spore Viability

2011 ◽  
Vol 77 (17) ◽  
pp. 6208-6214 ◽  
Author(s):  
I. Grand ◽  
M.-N. Bellon-Fontaine ◽  
J.-M. Herry ◽  
D. Hilaire ◽  
F.-X. Moriconi ◽  
...  
Keyword(s):  
Peracetic Acid ◽  
Laser Scanning ◽  
Sampling Procedure ◽  
Standard Test ◽  
Laser Scanning Microscopy ◽  
Fluorescence Labeling ◽  
Confocal Laser ◽  
Content Type ◽  
Surface Disinfection ◽  
Sampling Procedures

ABSTRACTThe standard test methods used to assess the efficiency of a disinfectant applied to surfaces are often based on counting the microbial survivors sampled in a liquid, but total cell removal from surfaces is seldom achieved. One might therefore wonder whether evaluations of microbial survivors in liquid-sampled cells are representative of the levels of survivors in whole populations. The present study was thus designed to determine the “damaged/undamaged” status induced by a peracetic acid disinfection forBacillus atrophaeusspores deposited on glass coupons directly on this substrate and to compare it to the status of spores collected in liquid by a sampling procedure. The method utilized to assess the viability of both surface-associated and liquid-sampled spores included fluorescence labeling with a combination of Syto 61 and Chemchrome V6 dyes and quantifications by analyzing the images acquired by confocal laser scanning microscopy. The principal result of the study was that the viability of spores sampled in the liquid was found to be poorer than that of surface-associated spores. For example, after 2 min of peracetic acid disinfection, less than 17% ± 5% of viable cells were detected among liquid-sampled cells compared to 79% ± 5% or 47% ± 4%, respectively, when the viability was evaluated on the surface after or without the sampling procedure. Moreover, assessments of the survivors collected in the liquid phase, evaluated using the microscopic method and standard plate counts, were well correlated. Evaluations based on the determination of survivors among the liquid-sampled cells can thus overestimate the efficiency of surface disinfection procedures.

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