scholarly journals Propidium iodide staining underestimates viability of adherent bacterial cells

2018 ◽  
Author(s):  
Merilin Rosenberg ◽  
Nuno F. Azevedo ◽  
Angela Ivask
Keyword(s):  
Dna Binding ◽  
Propidium Iodide ◽  
Laser Scanning Microscopy ◽  
Red Cells ◽  
Extracellular Dna ◽  
Dead Layer ◽  
Confocal Laser ◽  
Adherent Cells ◽  
Bacterial Viability ◽  
Viability Staining

AbstractCombining membrane impermeable DNA-binding stain propidium iodide (PI) with membrane-permeable DNA-binding counterstains is a widely used approach for bacterial viability staining. In this paper we show that PI staining of adherent cells in biofilms may significantly underestimate bacterial viability due to the presence of extracellular nucleic acids. We demonstrate that gram-positive Staphylococcus epidermidis and gram-negative Escherichia coli 24-hour initial biofilms on glass consist of 76 and 96% PI-positive red cells in situ, respectively, even though 68% the cells of either species in these aggregates are metabolically active. Furthermore, 82% of E. coli and 89% S. epidermidis are cultivable after harvesting. Confocal laser scanning microscopy (CLSM) revealed that this false dead layer of red cells is due to a subpopulation of double-stained cells that have green interiors under red coating layer which hints at extracellular DNA (eDNA) being stained outside intact membranes. Therefore, viability staining results of adherent cells should always be validated by an alternative method for estimating viability, preferably by cultivation.

scholarly journals Biodegradable Gene Carriers Containing Rigid Aromatic Linkage with Enhanced DNA Binding and Cell Uptake

Polymers ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 1080 ◽  
Author(s):  
Ju-Hui Zhang ◽  
Hui-Zhen Yang ◽  
Ji Zhang ◽  
Yan-Hong Liu ◽  
Xi He ◽  
...  
Keyword(s):  
Dna Binding ◽  
Laser Scanning ◽  
Transfection Efficiency ◽  
Laser Scanning Microscopy ◽  
Cell Uptake ◽  
Confocal Laser ◽  
Viral Gene ◽  
Cationic Polymers ◽  
Aromatic Rings ◽  
Gene Carriers

The linking and modification of low molecular weight cationic polymers (oligomers) has become an attracted strategy to construct non-viral gene carriers with good transfection efficiency and much reduced cytotoxicity. In this study, PEI 600 Da was linked by biodegradable bridges containing rigid aromatic rings. The introduction of aromatic rings enhanced the DNA-binding ability of the target polymers and also improved the stability of the formed polymer/DNA complexes. The biodegradable property and resulted DNA release were verified by enzyme stimulated gel electrophoresis experiment. These materials have lower molecular weights compared to PEI 25 kDa, but exhibited higher transfection efficiency, especially in the presence of serum. Flow cytometry and confocal laser scanning microscopy results indicate that the polymers with aromatic rings could induce higher cellular uptake. This strategy for the construction of non-viral gene vectors may be applied as an efficient and promising method for gene delivery.

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 Biofilm Formation by Streptococcus pneumoniae: Role of Choline, Extracellular DNA, and Capsular Polysaccharide in Microbial Accretion

2006 ◽  
Vol 188 (22) ◽  
pp. 7785-7795 ◽  
Author(s):  
Miriam Moscoso ◽  
Ernesto García ◽  
Rubens López
Keyword(s):  
Streptococcus Pneumoniae ◽  
Biofilm Formation ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Dimensional Structure ◽  
Confocal Laser ◽  
Upper Respiratory Tract ◽  
Teichoic Acids ◽  
Abiotic Surfaces ◽  
Biofilm Development

ABSTRACTStreptococcus pneumoniaecolonizes the human upper respiratory tract, and this asymptomatic colonization is known to precede pneumococcal disease. In this report, chemically defined and semisynthetic media were used to identify the initial steps of biofilm formation by pneumococcus during growth on abiotic surfaces such as polystyrene or glass. Unencapsulated pneumococci adhered to abiotic surfaces and formed a three-dimensional structure about 25 μm deep, as observed by confocal laser scanning microscopy and low-temperature scanning electron microscopy. Choline residues of cell wall teichoic acids were found to play a fundamental role in pneumococcal biofilm development. The role in biofilm formation of choline-binding proteins, which anchor to the teichoic acids of the cell envelope, was determined using unambiguously characterized mutants. The results showed that LytA amidase, LytC lysozyme, LytB glucosaminidase, CbpA adhesin, PcpA putative adhesin, and PspA (pneumococcal surface protein A) mutants had a decreased capacity to form biofilms, whereas no such reduction was observed in Pce phosphocholinesterase or CbpD putative amidase mutants. Moreover, encapsulated, clinical pneumococcal isolates were impaired in their capacity to form biofilms. In addition, a role for extracellular DNA and proteins in the establishment ofS. pneumoniaebiofilms was demonstrated. Taken together, these observations provide information on conditions that favor the sessile mode of growth byS. pneumoniae. The experimental approach described here should facilitate the study of bacterial genes that are required for biofilm formation. Those results, in turn, may provide insight into strategies to prevent pneumococcal colonization of its human host.

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.

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