scholarly journals Distribution of extracellular DNA in Listeria monocytogenes biofilm

2019 ◽  
Vol 37 (No. 6) ◽  
pp. 409-416
Author(s):  
Martina Šuláková ◽  
Jarmila Pazlarová ◽  
Rikke Louise Meyer ◽  
Kateřina Demnerová
Keyword(s):  
Listeria Monocytogenes ◽  
Laser Scanning ◽  
3D Structure ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Matrix Component ◽  
Signal Distribution ◽  
Culturing Conditions ◽  
Phylogenetic Lineages

Extracellular DNA (eDNA) is an abundant matrix component that protects biofilm from environmental stress, facilitate horizontal gene transfer, and serve as a source of nutrients. eDNA is also found in Listeria monocytogenes biofilm, but it is unknown to which extent its importance as a matrix component varies in terms of phylogenetic relatedness. This study aims to determine if these variations exist. Biofilm forming capacity of ten L. monocytogenes strains of different phylogenetic lineages and serotypes was examined using crystal violet assay at 37°C and 22°C. eDNA content was evaluated fluorometrically at 37°C and at 22°C, then the 3D structure of biofilm was studied by confocal laser scanning microscopy (CLSM). Biofilm forming capacity differed significantly between the culturing conditions and was higher at 37°C than at ambient temperature. eDNA signal distribution was found to be influenced by strain and lineage. CLSM images revealed information about spatial distribution in the biofilm. The information about the eDNA spatial organisation in the biofilm contributes to the understanding of the role of eDNA in a biofilm formation.

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.

Effects of Bacteriophage P100 at Different Concentrations on the Structural Parameters of Listeria monocytogenes Biofilms

2018 ◽  
Vol 81 (12) ◽  
pp. 2040-2044 ◽  
Author(s):  
CRISTINA RODRÍGUEZ-MELCÓN ◽  
ROSA CAPITA ◽  
CAMINO GARCÍA-FERNÁNDEZ ◽  
CARLOS ALONSO-CALLEJA
Keyword(s):  
Image Analysis ◽  
Listeria Monocytogenes ◽  
Laser Scanning ◽  
Digital Image Analysis ◽  
Surface Coverage ◽  
Structural Parameters ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Foodborne Diseases ◽  
Average Thickness

ABSTRACT Because listeriosis is one of the deadliest foodborne diseases, controlling and eradicating Listeria monocytogenes biofilms is a serious challenge for food safety. Biofilms (24 h old) formed on polystyrene by a L. monocytogenes strain of food origin were exposed for a further 24 h to 12 different concentrations (from 100 to 1011 PFU/mL) of the bacteriophage P100 (Listex P100). The structural parameters of biofilms were studied by using confocal laser scanning microscopy and digital image analysis. The biovolume in the observation field (14,121 μm2) of control (untreated) biofilms was 237,333.1 ± 2,692.6 μm3. The biomass of treated biofilms ranged from 164.7 ± 89.0 μm3 (biofilms exposed to 1010 PFU/mL) to 231,170.5 ± 15,142.0 μm3 (100 PFU/mL). The lowest biomass was achieved after treatment with 108 PFU/mL, with no further decrease in biovolume when higher phage concentrations were used. A strong (P &lt; 0.001) correlation was found between phage concentration (log units) and biovolume (−0.965), surface coverage (−0.939), roughness (0.976), maximum thickness (−0.853), and average thickness (−0.965). Findings from this research suggest that bacteriophage P100 at concentrations equal to or greater than 8 log PFU/mL successfully removes L. monocytogenes biofilms from polystyrene surfaces.

scholarly journals Autolysin-Independent DNA Release inStreptococcus pneumoniae in vitroBiofilms

2018 ◽  
Author(s):  
Mirian Domenech ◽  
Ernesto García
Keyword(s):  
Extracellular Matrix ◽  
Quorum Sensing ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Lytic Enzymes ◽  
Microbial Biofilms ◽  
Dna Release

ABSTRACTBiofilms are defined as layers of cells of microorganisms adhered to the surface of a substrate and embedded in an extracellular matrix and provide an appropriate environment for increased genetic exchange. Extracellular DNA (eDNA) is an essential component of the extracellular matrix of microbial biofilms, but the pathway(s) responsible for DNA release are largely unknown. Autolysis (either spontaneous or phage-induced) has been proposed the major event leading to the appearance of eDNA. The ‘suicidal tendency’ ofStreptococcus pneumoniaeis well-known, with lysis mainly caused by the triggering of LytA, the major autolytic amidase. However, the LytC lysozyme and CbpD (a possible murein hydrolase) have also been shown involved. The present work examines the relationship between eDNA, autolysins, and the formation and maintenance ofin vitropneumococcal biofilms, via fluorescent labelling combined with confocal laser scanning microscopy, plus genetic transformation experiments. Bacterial DNA release mechanisms other than those entailing lytic enzymes were shown to be involved by demonstrating that horizontal gene transfer in biofilms takes place even in the absence of detectable autolytic activity. It had been previously suggested that the quorum sensing systems ComABCDE and LuxS/AI-2 are involved in the production of eDNA as a response to the accumulation of quorum sensing signals, although our immunofluorescence results do not support this hypothesis. Evidence that the release of DNA is somehow linked to the production of extracellular vesicles byS. pneumoniaeis provided.

scholarly journals The exopolysaccharide–eDNA interaction modulates 3D architecture of Bacillus subtilis biofilm

2020 ◽  
Author(s):  
Na Peng ◽  
Peng Cai ◽  
Monika Mortimer ◽  
Yichao Wu ◽  
Chunhui Gao ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Titration Calorimetry ◽  
Confocal Laser ◽  
Physical Interaction ◽  
Matrix Components ◽  
Biofilm Development ◽  
Early Phases

Abstract Background Bacterial biofilms are a surface-adherent microbial community in which individual cells are surrounded by a self-produced extracellular matrix of polysaccharide, extracellular DNA (eDNA) and proteins. Interactions among matrix components within the biofilms are responsible for creating an adaptable structure during biofilm development. However, it is unclear how the interaction among matrix components contributes to the construction of the three-dimensional (3D) biofilm architecture. Results DNase I treatment could significantly inhibit B. subtilis biofilm formation in early phases. Confocal laser scanning microscopy (CLSM) and image analysis revealed that eDNA was cooperative with exopolysaccharide (EPS) in early stages of B. subtilis biofilm development, while EPS played a major structural role in the later stages. In addition, deletion of EPS production gene epsG in B. subtilis SBE1 resulted in loss of the interaction between EPS and eDNA, and reduction of biofilm biomass in pellicles at air-liquid interface. The physical interaction between these two essential biofilm matrix components was confirmed by isothermal titration calorimetry (ITC). Conclusions The biofilm 3D structures become interconnected through surrounding eDNA and EPS. eDNA interacts with EPS in the early phases of biofilm development, while EPS mainly participates in the maturation of biofilm. The findings of this study provide better understanding of the role of interaction between eDNA and EPS in shaping the biofilm 3D matrix structure and biofilm formation.

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 High Adhesion and Increased Cell Death Contribute to Strong Biofilm Formation in Klebsiella pneumoniae

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 277
Author(s):  
Siddhi Desai ◽  
Kinjal Sanghrajka ◽  
Devarshi Gajjar
Keyword(s):  
Cell Death ◽  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Bacterial Cells ◽  
High Adhesion ◽  
Biofilm Matrix

Klebsiella pneumoniae (Kp), is a frequent cause of hospital and community-acquired infections and WHO had declared it as a “priority pathogen”. Biofilm is a major virulence factor of Kp and yet the mechanism of strong biofilm formation in Kp is unclear. A key objective of the present study is to investigate the differences between strong and weak biofilms formed by clinical isolates of Kp on various catheters and in different media conditions and to identify constituents contributing to strong biofilm formation. Quantification of matrix components (extracellular DNA (eDNA), protein, exopolysaccharides (EPS), and bacterial cells), confocal laser scanning microscopy (CLSM), field emission gun scanning electron microscopy (FEG-SEM) and flow-cytometry analysis were performed to compare strong and weak biofilm matrix. Our results suggest increased biofilm formation on latex catheters compared to silicone and silicone-coated latex catheters. Higher amounts of eDNA, protein, EPS, and dead cells were observed in the strong biofilm of Kp. High adhesion capacity and cell death seem to play a major role in formation of strong Kp biofilms. The enhanced eDNA, EPS, and protein in the biofilm matrix appear as a consequence of increased cell death.

scholarly journals Subinhibitory Concentrations of Mupirocin Stimulate Staphylococcus aureus Biofilm Formation by Upregulating cidA

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Ye Jin ◽  
Yinjuan Guo ◽  
Qing Zhan ◽  
Yongpeng Shang ◽  
Di Qu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Multidrug Resistant ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Content Type ◽  
Subinhibitory Concentrations ◽  
Biofilm Development

ABSTRACT Previous studies have shown that the administration of antibiotics at subinhibitory concentrations stimulates biofilm formation by the majority of multidrug-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated the effect of subinhibitory concentrations of mupirocin on biofilm formation by the community-associated (CA) mupirocin-sensitive MRSA strain USA300 and the highly mupirocin-resistant clinical S. aureus SA01 to SA05 isolates. We found that mupirocin increased the ability of MRSA cells to attach to surfaces and form biofilms. Confocal laser scanning microscopy (CLSM) demonstrated that mupirocin treatment promoted thicker biofilm formation, which also correlated with the production of extracellular DNA (eDNA). Furthermore, quantitative real-time PCR (RT-qPCR) results revealed that this effect was largely due to the involvement of holin-like and antiholin-like proteins (encoded by the cidA gene), which are responsible for modulating cell death and lysis during biofilm development. We found that cidA expression levels significantly increased by 6.05- to 35.52-fold (P < 0.01) after mupirocin administration. We generated a cidA-deficient mutant of the USA300 S. aureus strain. Exposure of the ΔcidA mutant to mupirocin did not result in thicker biofilm formation than that in the parent strain. We therefore hypothesize that the mupirocin-induced stimulation of S. aureus biofilm formation may involve the upregulation of cidA.

scholarly journals Sanguinarine Inhibits Mono- and Dual-Species Biofilm Formation by Candida albicans and Staphylococcus aureus and Induces Mature Hypha Transition of C. albicans

2020 ◽  
Vol 13 (1) ◽  
pp. 13 ◽  
Author(s):  
Weidong Qian ◽  
Wenjing Wang ◽  
Jianing Zhang ◽  
Miao Liu ◽  
Yuting Fu ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Inhibitory Concentration ◽  
Fluorescent Dyes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Matrix Component ◽  
New Agents ◽  
Diffusion Assay ◽  
And Staphylococcus Aureus

Previous studies have reported that sanguinarine possesses inhibitory activities against several microorganisms, but its effects on mono- and dual-species biofilms of C. albicans and S. aureus have not been fully elucidated. In this study, we aimed to evaluate the efficacy of sanguinarine for mono- and dual-species biofilms and explore its ability to induce the hypha-to-yeast transition of C. albicans. The results showed that the minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC90) of sanguinarine against C. albicans and S. aureus mono-species biofilms was 4, and 2 μg/mL, respectively, while the MIC and MBIC90 of sanguinarine against dual-species biofilms was 8, and 4 μg/mL, respectively. The decrease in the levels of matrix component and tolerance to antibiotics of sanguinarine-treated mono- and dual-species biofilms was revealed by confocal laser scanning microscopy combined with fluorescent dyes, and the gatifloxacin diffusion assay, respectively. Meanwhile, sanguinarine at 128 and 256 μg/mL could efficiently eradicate the preformed 24-h biofilms by mono- and dual-species, respectively. Moreover, sanguinarine at 8 μg/mL could result in the transition of C. albicans from the mature hypha form to the unicellular yeast form. Hence, this study provides useful information for the development of new agents to combat mono- and dual-species biofilm-associated infections, caused by C. albicans and S. aureus.

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 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.

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