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Extracellular Polymeric Substance Matrix

Extracellular Polymeric Substance Matrix: A complex, hydrated network of polysaccharides, proteins, and other macromolecules secreted by microorganisms.
This matrix provides structural integrity, facilitates intercellular communication, and plays a crucial role in the formation of biofilms.
Understanding the properties and functionailty of this extracellular matrix is key to advancing research in areas such as microbial ecology, bioremediation, and biomedicine.

Most cited protocols related to «Extracellular Polymeric Substance Matrix»

1
Evaluation of Candida albicans Biofilm Formation
Three C. albicans strains were chosen for their varied abilities to form biofilms. The reference strain DAY185 produces extensive biofilms with abundant extracellular matrices and variable cell morphologies [19 (link)]. The two mutant strains lack adhesions, resulting in a partial biofilm defect for als3Δ/Δ and a more extensive biofilm defect in strain als1Δ/Δ als3Δ/Δ [8 ,20 (link)–22 (link)]. To evaluate biofilm formation, overnight cultures grown in yeast peptone dextrose (YPD) broth [8 ] at 30°C with orbital shaking at 200 rpm were enumerated by hemocytometer. Cells were resuspended in RPMI-MOPS [7 (link)] at 106 cells/ml and each well of a six-well polystyrene plate was inoculated with 1 ml of this suspension. After a 1 h adhesion period, the inoculum was removed and fresh media was applied. Biofilms were grown for 48 h at 37°C on an orbital shaker at 50 rpm. For comparative scanning electron microscopy (SEM), biofilms were formed on coverslips and processed and examined as previously described [23 (link)].
Protocols for the XTT and crystal violet assays have been described [7 (link),24 (link)]. For the crystal violet assay, 4% aqueous crystal violet was added for 45 min and 100 µl aliquots were taken from each well for absorbance measurement (595 nm). Candida DNA quantification was accomplished using a commercially available kit (Genomic DNA Wizard Kit – Promega) following cellular disruption by bead-beating [25 (link)]. DNA samples were also used for qPCR with ACT1 primers and probes [23 (link)] and Quantitect Probe qPCR kit (Qiagen) performed with the CFX96 Real-Time PCR Detection System (Bio-Rad). The Protein Assay Kit (Pierce) was used to determine total cellular protein after biofilms were scraped from wells into 1 ml of ddH2O, sonicated for 20 min, disrupted by bead-beating, and boiled. Dry cell weight of each biofilm was determined following disruption, collection, and then dehydrated by vacuum centrifugation. Viable burdens were determined by plating serial dilutions following biofilm disruption, vortexing, and sonicated using a waterbath for 20 min to disperse cells [14 ].
Reproducibility was estimated by the standard deviation and coefficient of variation among replicates. At least six biofilms were grown for each strain and assay.
Taff H.T., Nett J.E, & Andes D.R. (2011). Comparative analysis of Candida biofilm quantitation assays. Medical Mycology, 50(2), 214-218.
Publication 2011
Biofilms Biological Assay Candida Cells Centrifugation Extracellular Polymeric Substance Matrix Genome Glucose morpholinopropane sulfonic acid Oligonucleotide Primers Peptones Polystyrenes Promega Proteins Scanning Electron Microscopy Strains Technique, Dilution Vacuum Violet, Gentian Yeast, Dried
2
Ultrastructural Analysis of Biofilms
Before proceeding with the ultrastructural analysis of S. wiggsiae, we tested conventional SEM and VP-SEM protocols on S. mutans’ biofilm (Figure 1 and Figure 2), but we were unsatisfied by the results, so we decided to test an original preparation procedure, OsO4-RR-TA-IL, (Figure 3), never reported before in the literature, adopting osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA) impregnation and ionic liquid (IL) drop casting instead of sputter coating. We developed this procedure to combine the advantages of the conventional SEM protocol (image quality, magnification, resolution and long resistance under electron beam) with the advantages of VP-SEM, i.e., reduced preparation time (few steps protocol), minimal sample loss and actual sample structure preservation (achieved avoiding dehydration and drying). To obtain these requirements, the protocol should keep the samples hydrated and not require drying or sputter coating; should allow observation under high-vacuum conditions at acceleration voltages of 1520 kV. High magnifications and high-resolution images obtained under these conditions would allow an ultrastructural characterization without artifacts. To this aim, we decided to use a combination of OsO4-RR-TA, reagents already used for a long time in electron microscopy and IL, each one characterized by its own peculiar properties.
RR is a polycationic dye generally used in post-fixation steps, together with osmium or TA. It preserves integrity of negatively charged complex carbohydrates. It is useful to avoid polysaccharide loss in conventional SEM protocols (it is usually high up to 40%) [30 (link)]. RR cationic properties enable preservation of EPS polysaccharides and creates electrostatic or ionic links with EPS components, stabilizing the biofilm matrix and avoiding sample shrinking [23 (link),26 ,30 (link),31 (link),32 (link),33 (link),34 (link),35 (link)]. RR for the visualization of extracellular structures with EM was pioneered by Luft [36 ] and then used on Staphylococcus aureus [26 ,37 (link),38 (link)], Pseudomonas spp. [39 ], Enterococcus faecalis [34 (link)] and Klebsiella pneumoniae [34 (link)].
Tannic acid reacts with osmium tetroxide and increases lipid retention, forming complexes that link to proteins and carbohydrates [40 ]. Consequently, they enhance extracellular matrix resistance to mechanical damage during preparation procedures, thanks to a sort of specimen hardening [41 (link),42 (link),43 (link),44 (link)]. This method renders the sample itself conductive (not only its surface, as it happens with sputter coating), enhances the image contrast without charging phenomena and allows a three-dimensional observation of its sub-surface structures under higher voltages in comparison with VP-SEM [45 ].
Ionic liquids are, at room temperature, molten salts with high electronic conductivity and irrelevant vapor pressure [46 ,47 (link)]. These properties allow their use in SEM as a substitute for metal coating [48 (link),49 (link)]. We covered biofilm samples with IL, to maintain them wet during the SEM investigation. Even under high-vacuum conditions, ILs resist evaporation, and their use eliminates biofilm dehydration, critical point drying and sputter coating, which contributes to sample preservation.
OsO4-RR-TA-IL was evaluated as the most suitable protocol on S. mutans (Figure 3, Table 1 and Table 2), so we used the same to characterize S. wiggsiae and its biofilm’s ultrastructural architecture (Figure 4 and Figure 5).
Samples of S. mutans grown on aluminium disks were processed as reported in Table 1. Samples of S. wiggsiae’s biofilm grown for 120 h on bioactive glass discs were processed (after evaluation of results on S. mutans) with the OsO4-RR-TA-IL protocol. In order to provide accurate measurements of bacterial cell dimensions, we randomly selected several images from 20000× to 35000×. To determine bacterial cell length, we measured, in each selected image, only bacterial cells longitudinally arranged in which both extremities were fully visible (for an overall amount of 100 bacterial cells). To measure the bacterial cell diameter, we used the same images, but we considered only cells with one pole perpendicular to the surface. Once again, we measured 100 bacterial cells. Measurements were carried on by the Image J software and by the SEM image analysis software Hitachi Map 3D (Digital Surf, France). Measure values were statistically analyzed by the MedCalc © software.
Bossù M., Selan L., Artini M., Relucenti M., Familiari G., Papa R., Vrenna G., Spigaglia P., Barbanti F., Salucci A., Di Giorgio G., Rau J.V, & Polimeni A. (2020). Characterization of Scardovia wiggsiae Biofilm by Original Scanning Electron Microscopy Protocol. Microorganisms, 8(6), 807.
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Publication 2020
Acceleration Aluminum Bacteria Biofilms Biologic Preservation Carbohydrates Cations Cells Dehydration Electric Conductivity Electron Microscopy Electrons Electrostatics Enterococcus faecalis Extracellular Matrix Extracellular Polymeric Substance Matrix Fertilization Fever Fingers Ionic Liquids Ions Klebsiella pneumoniae link protein Lipids Metals Osmium Osmium Tetroxide Polar Bodies polycations Polysaccharides Pseudomonas Retention (Psychology) Salts Sclerosis Staphylococcus aureus Infection Tannins Vacuum Vapor Pressure VP protocol
3
Biofilm Formation and Characterization of MRSA and PA
Single- or multi-species in vitro biofilm growth of MRSA and PA was established in 24-well (flat-bottom) polystyrene tissue culture plates (BD Falcon, Sparks, MD, USA) using a static model and a previously described procedure (Christensen et al., 1985 (link); Yadav et al., 2015b (link)). The biofilm biomass was quantified using a crystal violet (CV) microtiter plate assay, and the bacterial loads within biofilms were enumerated by colony forming unit (CFU) counts. MRSA or PA cell suspensions (1 × 107), individually or in combination in TSB media, were inoculated (1 mL) in 24-well polystyrene plates. The plates were incubated at 37°C for 24 h. After incubation, medium was discarded, and plates were gently washed with 1 mL sterile water. Thereafter, plates were air-dried and stained with 200 μL CV (0.1%) for 15 min. Excess stain was decanted, and plates were washed three times with sterile distilled water. The biofilm was dissolved in 1 mL (95%) ethanol and the optical density (OD) at 570 nm was measured in an automatic spectrophotometer. All experiments were performed in triplicate and the average was calculated. The experiments were repeated three times.
Alternatively, MRSA, PA, or combinations of both species were grown in TSB medium under the same conditions. CFUs were counted to quantify the number of viable cells growing in the biofilms. Biofilms were dissolved with sonication at 50 W for 10 s, serially diluted, and plated on selective medium, specifically ORSAB or PAB with CN supplement, to determine CFU-values.
To characterize the biofilm matrix, 24-h pre-established biofilms of MRSA or PA were treated with 10 mM sodium metaperiodate (Sigma, St. Louis, MO, USA), 100 μg/mL DNase I (Roche, Mannheim, Germany), 100 μg/mL alginate lyase (sigma), and 100 μg/mL proteinase K (sigma) by procedure previously described (Gutiérrez et al., 2014 (link)). The control biofilms were treated with respective buffer. The biofilms biomass was quantified as described above.
Yadav M.K., Chae S.W., Go Y.Y., Im G.J, & Song J.J. (2017). In vitro Multi-Species Biofilms of Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa and Their Host Interaction during In vivo Colonization of an Otitis Media Rat Model. Frontiers in Cellular and Infection Microbiology, 7, 125.
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Publication 2017
Biofilms Biological Assay Buffers Cells Deoxyribonucleases Dietary Supplements Endopeptidase K Ethanol Extracellular Polymeric Substance Matrix Methicillin-Resistant Staphylococcus aureus poly(beta-D-mannuronate) lyase Polystyrenes sodium metaperiodate Stains Sterility, Reproductive Tissues Violet, Gentian Vision
4
Candida albicans Biofilm Matrix Isolation
A rolling bottle system was used to generate matrix for analyses. Briefly, aliquots of C. albicans grown in RPMI (RPMI 1640 buffered with morpholinepropanesulfonic acid [MOPS]) were used to inoculate a polystyrene roller bottle with a surface area of 850 cm2 (Corning product number 3970). Bottles were placed on a roller apparatus (Wheaton Science Products, Millville, NJ), rolling at the rate of 20 rpm at 37°C. After 24 h, the biofilm culture medium was replaced and the bottles were incubated for another 24 h. After medium removal, the C. albicans biofilms were dislodged by spatula and gently sonicated to avoid cell wall disruption (42 kHz for 20 min [Branson 1510 Ultrasonic Cleaner sonicator] followed by sonication with a 1-cm by 5-cm probe in an Intrasonic Processor [Cole Parmer, Vernon Hills, IL] at an amplitude of 70 for 10 min). The aggregate biofilm was then centrifuged to separate fungal cells and matrix. The supernatant-containing matrix was then collected and lyophilized. The sample was resuspended in water and dialyzed (molecular weight cutoff of 3) for 5 consecutive days and again lyophilized yielding the “crude” biofilm matrix. Overall, a total of 700 bottles of the matrix corresponding to the biofilm area of 59.5 m2 were collected for analysis. A similar cell mass of planktonic (nonbiofilm) C. albicans was collected and similarly processed to discern the impact of matrix processing on the cell wall.
Zarnowski R., Westler W.M., Lacmbouh G.A., Marita J.M., Bothe J.R., Bernhardt J., Lounes-Hadj Sahraoui A., Fontaine J., Sanchez H., Hatfield R.D., Ntambi J.M., Nett J.E., Mitchell A.P, & Andes D.R. (2014). Novel Entries in a Fungal Biofilm Matrix Encyclopedia. mBio, 5(4), e01333-14.
Publication 2014
Acids Biofilms Candida albicans Cells Cell Wall Extracellular Polymeric Substance Matrix Plankton Polystyrenes Ultrasonics
5
Evaluation of Peptide Effects on Bacterial Biofilm
Bacterial biofilm was grown on glass cover slips in 24-well plates in 0.5X MHB in static conditions. In particular, bacterial cells from an overnight culture were diluted to about 1 × 108 CFU/mL and then seeded into wells for 4 or 24 h at 37 °C in the presence of the peptide under test, in order to evaluate biofilm attachment and formation, respectively. When effects on preformed biofilm were evaluated, bacterial biofilms were formed for 24 h at 37 °C, and then treated with peptides under test for further 24 h to evaluate their ability to eradicate preformed biofilm. Afterwards, non-adherent bacteria were removed by gently washing samples with sterile phosphate buffer and viability of cells embedded into biofilm structure was determined by sample staining with LIVE/DEAD® BacLight™ Bacterial Viability kit (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA), while FilmTracer™ SYPRO® Ruby biofilm matrix dye has been used to stain matrices of biofilms (Invitrogen, Carlsbad, CA, USA). Staining was performed accordingly to manufacturer instructions. Biofilm images were captured by using a confocal laser scanning microscopy (Zeiss LSM 710, Zeiss, Germany) and a 63X objective oil immersion system. Biofilm architecture was analyzed by using the Zen Lite 2.3 software package (Zeiss, Germany). Each experiment was performed in triplicate. All images were taken under identical conditions.
Gaglione R., Cesaro A., Dell’Olmo E., Di Girolamo R., Tartaglione L., Pizzo E, & Arciello A. (2020). Cryptides Identified in Human Apolipoprotein B as New Weapons to Fight Antibiotic Resistance in Cystic Fibrosis Disease. International Journal of Molecular Sciences, 21(6), 2049.
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Publication 2020
Bacteria Bacterial Viability Biofilms Buffers Cell Culture Techniques Cell Survival Extracellular Polymeric Substance Matrix Immersion Microscopy, Confocal, Laser Scanning Molecular Probes Peptides Phosphates Stains Sterility, Reproductive Sypro Ruby

Most recents protocols related to «Extracellular Polymeric Substance Matrix»

1
Dictyostelium Strains Suppress Soft Rot Pathogens
Not available on PMC !

Example 1

119 Dicty strains were screened for their ability to feed on Dickeya (Dd) or Pectobacterium (Pcc) at 10° C. This assay was performed by inoculating Dd or Pcc on a low nutrient medium (SM2 agar) that supports both bacterial and Dicty growth. Dicty spores from individual strains were then inoculated on top of the bacterial growth and incubated at 10° C. to mimic potato storage temperatures. Dicty strains that successfully fed on Dd or Pcc created visible clearings in the lawn of bacterial growth and ultimately produced sporangia (fruiting bodies) that rose from the agar surface. An example of the phenotype that was considered successful clearing of bacteria is shown in FIG. 3A. From this initial screen, 36 Dicty strains that were capable of feeding on both Dd and Pcc at 10° C. were identified (FIG. 1B).

Of the 36 strains capable of feeding on both Dd and Pcc, 34 came from the Group 4 Dictyostelids (FIG. 1). This group includes D. discoideum, D. giganteum, D. minutum, D. mucoroides, D. purpureum, and D. sphaerocephalum (72). The results indicate that this group is particularly enriched in Dd and Pcc-feeding strains.

A further experiment was performed to identify Dicty species capable of feeding on biofilms of Dd and Pcc. Microporous polycarbonate membranes (MPMs) are widely reported to support biofilm formation of numerous Enterobacteriaceae species (2, 63, 70, 71). It was determined if Dd and Pcc formed biofilms on MPMs and determined if Dicty strains were capable of feeding on these biofilms. Membranes were placed on top of SM2 agar to provide Dd and Pcc with nutrients for growth. Bacteria were then inoculated on the surface of the MPMs and growth was monitored over the course of 1 week by washing bacteria off the membranes and performing dilution plating for colony counting. Growth of both bacterial strains plateaued around 4 dpi (FIG. 2).

From these results, it was determined that the best time to collect inoculated MPMs for biofilm analysis was at 2 dpi. Scanning electron microscopy (SEM) is commonly used to confirm biofilm formation by detecting extracellular polymeric substance (EPS) that forms the biofilm matrix (2). Samples of Dd and Pcc after 2 days of growth on MPMs in the presence and absence of Dicty are analyzed using SEM.

19 Dicty strains identified as active were tested for their ability to feed on Dd and Pcc growing on MPMs. These experiments were performed by establishing Dd and Pcc growth on MPMs overlaid on SM2 agar at 37° C. for 24 hr. Dicty spores were then applied to the center of bacterial growth in a 5 uL drop containing 1000 spores. Bacteria and Dicty were incubated at 10° C. for 2 weeks before remaining bacteria were washed off and colonies were counted. Representative images of Dicty growing on Dd and Pcc on MPMs are shown in FIG. 3A.

No Dicty strains produced a statistically significant reduction in Dd viability compared to the non-treated control. However, treating Dd lawns with Cohen 36, Cohen 9, WS-15, WS-20, and WS-69 consistently reduced the number of viable bacteria by approximately 100,000-fold compared to the non-treated control (FIG. 3B). Cohen 9 was the only Dicty strain that produced a statistically significant reduction in viability of Pcc compared to the non-treated control (FIG. 3C). Other Dicty strains capable of reducing the number of viable Pcc by at least 100,000-fold were Cohen 35, Cohen 36, WS-647, and WS-69 (FIG. 3C).

It was observed that Dicty strains Cohen 9, Cohen 36, and WS-69 were capable of feeding on both Dd and Pcc when these bacteria were cultured on SM2 agar and MPMs (FIGS. 1 and 3). These strains were also particularly effective feeders as all three reduced the number of viable Dd and Pcc on MPMs at 10° C. by 100,000-fold compared to the non-treated control (FIGS. 3B and 3C).

To determine if these strains could suppress soft rot development on seed potato tubers, tubers were tab-inoculated with Dd or Pcc and treated with spores from each Dicty strain. Seed potatoes were surface-sterilized and punctured using a sterile screw to a depth of 1.5 mm. Overnight cultures of Dd and Pcc were suspended in 10 mM potassium phosphate buffer, diluted to an OD600 of approximately 0.003, and administered as a 5 μL drop into the wound. Next, 5 of a Dicty spore suspension (100,000 spores) was added to the wound. Inoculated seed potatoes were placed in a plastic container with moist paper towels and were misted with water twice a day to maintain a high humidity. After 3 days at room temperature, seed potatoes were sliced in half and the area of macerated tissue was quantified using ImageJ.

All three strains reduced the severity of soft rot caused by Dd and Pcc (FIG. 4). Cohen 36 was the most effective strain on both Dd and Pcc: reducing the area of tissue maceration by 60% and 35%, respectively (FIG. 4B). Treating seed potatoes with WS-69 reduced the area of tissue maceration by 50% and 30% for Dd and Pcc, respectively (FIG. 4B). Finally, Cohen 9 was the least effective, but still able to reduce tissue maceration caused by Dd and Pcc by 25% and 20%, respectively (FIG. 4B).

FIG. 7 shows that three Dicty isolates control Dd and Pcc in seed tubers (at 25° C.). Two sets of data from different weeks were normalized to the Dickeya or Pectobacterium only bacterial control. The average area of macerated potato tissue measured in mm2 was set as “1” or “100%”. The average of all the other treatments including Dicty were divided by bacteria only control and multiplied by 100 to obtain a percentage. Each set contained 5 tubers per treatment.

Dicty should be capable of sporulating at temperatures as cold as 10° C. on a potato surface if they are applied as a one-time pre-planting or post-harvest treatment. Sporulation was assessed by inoculating small potato discs (5×6 mm) with 10 μL of Dd or Pcc suspensions at an OD600 of 3×10−5 and Dicty spores at a concentration of 1×107 spores/mL. Potato discs were kept in a covered 96-well plate for two weeks at 10° C. followed by visual inspection for son using a dissecting microscope. Representative images of a strain producing many sori (WS-517) and a strain producing few sori (WS-69) are shown in FIG. 5. Of the 11 strains evaluated, only Cohen 9 and WS-20 were unable to sporulate in the presence of both pathogens (Table 1).

TABLE 1
Assessment of Dicty sporulation at 10° C. on potato
in the presence of Dd or Pcc. A (✓) indicates sori
have been observed while a ( [Figure (not displayed)]  ) means they have not.
Dicty strainDdPcc
Cohen 9✓[Figure (not displayed)]
Cohen 36✓✓
WS-69✓✓
WS-517✓✓
WS-588✓✓
WS-606✓✓
WS-15✓✓
WS-20[Figure (not displayed)] ✓
DC-7✓✓
DC-61✓✓
WS-116d✓✓

Example 2

This example describes the use of a high throughput screening assay to identify Dicty strains from Alaska (e.g., BAC10A, BAF6A, BAC3A, NW2, KB4A (ATCC® MYA-4262™) SO8B, SO3A, BAF9B, IC2A (ATCC® MYA-4259™), AK1A1 (ATCC® MYA-4272™) PBF4B (ATCC® MYA-4263), PBF8B, BSB1A, SO5B (ATCC® MYA-4249), PBF3C, PBF6B, NW2B, NW10B (ATCC® MYA-4271™), PBF9A, IC5A (ATCC® MYA-4256TH), ABC8A (ATCC® MYA-4260), NW16B, ABC10B, ABB6B (ATCC® MYA-4261), BA4A (ATCC® MYA-4252), AKK5A, AKK52C, HP4 (ATCC® MYA-4286), HP8 (ATCC® MYA-4284), or NW9A) that feed on Dd and Pcc at 10° C. on potatoes.

Results from 11 Dicty strains screened against Dd at 10° C. are presented in FIG. 6. Data was analyzed for significance using a one-way analysis of variance (ANOVA; alpha =0.05) with Tukey's honest significant difference (HSD) test to compare means between the treatments and the No Dicty control. A reduction in Dd proliferation when potato discs were treated with Dicty strains Cohen 9, Cohen 36, WS-15, Maryland 18a, BAF6A, NW2, and SO3A.

The Alaskan Dicty strains, and those identified in Example 1, are further tested against coinfections of Dd and Pcc. It is useful to identify Dicty strains that can suppress Dd and Pcc coinfections as these two pathogens have been isolated together from diseased potatoes (15). The ability of Dicty strains with different feeding preferences (Dd vs. Pcc) to complement each other when administered as a cotreatment is assayed.

US11864559B2. Therapeutic amoeba and uses thereof (2024-01-09). AmebaGone, LLC [US]. Inventors: Marcin Filutowicz [US], Ryan Kessens [US], Amy Jancewicz [US].
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Patent 2024
A-A-1 antibiotic Agar Amoeba Bacteria Biofilms Buffers Coinfection Cold Temperature Combined Modality Therapy Dickeya Dictyosteliida Enterobacteriaceae Extracellular Polymeric Substance Matrix Extracellular Polymeric Substances High-Throughput Screening Assays Human Body Humidity Microscopy neuro-oncological ventral antigen 2, human Nutrients Pathogenicity Pectobacterium Phenotype Plant Tubers polycarbonate potassium phosphate Scanning Electron Microscopy Solanum tuberosum Sporangia Spores Sterility, Reproductive Strains Technique, Dilution Tissue, Membrane Tissues Wounds
2
Extraction and Purification of Exopolysaccharides from Geobacillus
The Geobacillus sp. strain WSUCF1 media was prepared in a 1000 mL flask containing 6 g of shredded corn stover, 20 g yeast extract, 3 g NaCl, and 1000 mL of distilled (DI) water [9 (link)]. The corn stover, yeast extract, and NaCl solution was first made homogeneous using a magnetic stir bar with the pH set to 7.0, then allowed to autoclave at 121 °C for 20 min. Once the media had cooled, the flask was inoculated with the WSUCF1 bacteria and placed in a shaker set at 60 °C for 24 h. After 24 h, the liquid culture was passed through a Büchner funnel with the bottom of the funnel lined with a 0.22 µm pore size Whatman paper filter to separate the corn stover from the culture.
The bacterial cells were separated from the biofilm matrix-containing supernatant, which includes the EPS, via centrifugation at 8000× g for 20 min in 50 mL tubes or sometimes 500 mL centrifuge bottles. To separate the media from the EPS further, an optional step involves allowing the supernatant to evaporate via rotary evaporator until approximately 250–300 mL of water was removed. To precipitate the crude EPS, a 1:1 ratio of absolute ethanol was added to the remaining supernatant and allowed to sit in a freezer overnight at −20 °C. Lastly, the crude EPS was obtained by centrifuging the ethanol/supernatant solution at 8000× g for 40 min [9 (link)]. Following collection, the crude EPS was stored at −20 °C without a cryoprotectant.
Laubach J.M, & Sani R.K. (2023). Thermophilic Exopolysaccharide Films: A Potential Device for Local Antibiotic Delivery. Pharmaceutics, 15(2), 557.
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Publication 2023
Bacteria Cells Centrifugation Cryoprotective Agents Ethanol Extracellular Polymeric Substance Matrix Geobacillus Maize Sodium Chloride Suby's G solution Yeast, Dried
3
Quantitative Biofilm Assay for CRKP
The biofilm production and hypermucoviscous (HMV) phenotype are critical causes for CRKP colonization and persistence in the host. Biofilm production is important to the virulence of K. pneumoniae because the biofilm matrix promotes the relocation of antibiotic-resistance mobile elements while physically defending bacteria, thus enlarging microbial endurance to antibiotics, bacterial perseverance, and distribution [26 (link)–28 (link)]. Biofilm destruction needs high antimicrobial concentrations, which are often unbearable to succeed due to drug-related toxicity. Thus, regress is common even after targeted and extended therapies. According to these explanations, selecting bacterial isolates that can produce biofilm is essential for treatment [26 (link)]. Biofilm was determined using a quantitative microtiter plate (MTP) method [29 ]. Last, the microplate was evaluated using a microplate reader at 570 nm. The cut-off value for optical density (O.D.) was assigned. Each test was performed three times.
Mohammadi M., Saffari M., Siadat S.D., Hejazi S.H., Shayestehpour M., Motallebi M, & Eidi M. (2023). Isolation, characterization, therapeutic potency, and genomic analysis of a novel bacteriophage vB_KshKPC-M against carbapenemase-producing Klebsiella pneumoniae strains (CRKP) isolated from Ventilator-associated pneumoniae (VAP) infection of COVID-19 patients. Annals of Clinical Microbiology and Antimicrobials, 22, 18.
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Publication 2023
Antibiotic Resistance, Microbial Antibiotics, Antitubercular Bacteria Biofilms Extracellular Polymeric Substance Matrix Klebsiella pneumoniae Microbicides Phenotype Toxicity, Drug Virulence
4
Biofilm Reduction by Peptides and Ciprofloxacin
To investigate the biofilm-reductive effects of peptides and ciprofloxacin, the biofilm of DRPa-4009 cells was formed on the plastic coverslips for 48 h. After treatments of peptides (64 µM) and ciprofloxacin (256 µM) for 24 h, the coverslips were washed with SP buffer and stained with FilmTracer SYPRO Ruby biofilm matrix stain (Thermo Fisher Scientific Co., Ltd., Seoul, Korea) according to the manufacturer’s protocol. Samples were analyzed by CLSM (A1R HD 25, Nikon, Japan) [30 (link),31 (link)].
Kim Y.M., Son H., Park S.C., Lee J.K., Jang M.K, & Lee J.R. (2023). Anti-Biofilm Effects of Rationally Designed Peptides against Planktonic Cells and Pre-Formed Biofilm of Pseudomonas aeruginosa. Antibiotics, 12(2), 349.
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Publication 2023
Aftercare Biofilms Buffers Ciprofloxacin Extracellular Polymeric Substance Matrix Peptides Stains Sypro Ruby
5
Antisense-acting Riboswitch Regulates Transcriptional Regulator
We identified an antisense-acting c-di-GMP-I riboswitch [51 (link)] regulating the paen-P40081_02330 gene coding a transcriptional regulator of the XRE family in the Paenibacillus sp. FSL P4 genome (Fig 2E). This transcriptional regulator, whose nearest homolog is SinR, a transcriptional repressor known as the master regulator for biofilm formation in Bacillus subtilis, controls the expression of genes involved in exopolysaccharide, matrix protein production, and motility.
The synthesis of signaling molecules is one strategy bacteria employ to sense alterations in their environment in order to rapidly adjust according to those changes. One of these molecules is the bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP), which is able to induce the transition from a unicellular motile state to a multicellular sessile state through phosphorelay systems that control biofilm and spore formation (Fig 2E) [52 (link)–55 (link)].
For many bacteria, the quorum-sensing intercellular communication mechanism is essential for gene expression pattern coordination at the population level. Furthermore, for motility, biofilm matrix production, and spore formation processes, quorum sensing is critical, as it is one of the main strategies to sense and respond to environmental changes (Fig 2E) [56 (link)–59 (link)].
The c-di-GMP-I antisense-acting riboswitch regulation of a transcription factor coded by the paen-P40081_02330 gene that we propose is based on the detection of this molecule, triggering the expression of the genes involved in biofilm and spore formation, as well as quorum-sensing processes, and having a general effect on their expression when it is not sensed, thanks to its close relationship with the master regulator of these processes, Spo0A (Fig 2E).
As mentioned above, in the literature, there are only two examples of the regulation of gene expression mediated by riboswitches that act in an opposite direction, one of which is the cobalamin antisense-acting riboswitch identified at the 3`end of the gene encoding the PocR transcriptional regulator of the enzymes involved in the metabolism of propanediol in L. monocytogenes, the enzymes of which require cobalamin as a cofactor to be active. In our study, we identified cobalamin antisense-acting riboswitches in the orthologous pocR genes of other Listerias and in the Enterococcaceae Vagococcus carniphilus. The regulation of genes encoding transcription factors (TFs) makes it possible to extend the regulatory outcome of the riboswitches that act in an opposite direction to multiple functionally related genes in different operons, which have in common the operator site of the corresponding TFs. In our study, the c-di-GMP-I, cobalamin, and TPP antisense-acting riboswitches were the most frequent riboswitches of this group.
Serrano-Gutiérrez M, & Merino E. (2023). Antisense-acting riboswitches: A poorly characterized yet important model of transcriptional regulation in prokaryotic organisms. PLOS ONE, 18(2), e0281744.
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Publication 2023
Bacillus subtilis Bacteria Biofilms Cyclic GMP Enterococcaceae Enzymes Extracellular Polymeric Substance Matrix factor A Gene Expression Gene Expression Regulation Genes Genes, Essential Genome Listeria Metabolism Motility, Cell Multiple Birth Offspring Operon Paenibacillus Propylene Glycol Proteins Riboswitch Spores Transcription, Genetic Transcription Factor Vagococcus carniphilus Vitamin B12

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Filmtracer sypro ruby biofilm matrix stain by Thermo Fisher Scientific
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FilmTracer SYPRO Ruby Biofilm Matrix Stain is a fluorescent stain used for the detection and visualization of extracellular polymeric substances (EPS) in biofilm samples. It binds non-specifically to proteins within the biofilm matrix, allowing for the observation of the biofilm structure under fluorescent microscopy.
2
Syto 9 green fluorescent nucleic acid stain by Thermo Fisher Scientific
Sourced in United States, Spain, Australia
SYTO 9 Green Fluorescent Nucleic Acid Stain is a DNA-binding dye. It can be used to stain and detect nucleic acids in various biological samples.
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Ruthenium red by Merck Group
Sourced in United States, Germany, India, United Kingdom, France
Ruthenium red is a coordination complex of ruthenium with the molecular formula Ru(III)Cl3(NH3)5. It is a dark reddish-brown crystalline powder that is soluble in water and alcohol. Ruthenium red is commonly used as a stain in electron microscopy and histology due to its ability to bind to certain biological molecules, such as acidic polysaccharides and glycosaminoglycans.
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Alexa fluor 647 labeled dextran conjugate by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 647-labeled dextran conjugate is a fluorescent-labeled dextran compound. It consists of the Alexa Fluor 647 dye molecule covalently attached to dextran, a polysaccharide. This product is designed for use in various biological and biomedical research applications that require fluorescent labeling of dextran.
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Proteinase k by Merck Group
Sourced in United States, Germany, United Kingdom, Italy, Japan, Sao Tome and Principe, Canada, Belgium, Macao, Australia, Israel, France, China, Czechia, Singapore, Norway, Austria, Switzerland, Spain
Proteinase K is a serine protease enzyme that is commonly used in molecular biology and biochemistry laboratories. It is a highly active enzyme that efficiently digests a wide range of proteins, including those found in cell membranes, cytoplasmic proteins, and nuclear proteins. Proteinase K is known for its ability to effectively inactivate DNases and RNases, making it a valuable tool for the purification and isolation of nucleic acids.
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Syto9 by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China, France, Germany, India, Japan, Australia
SYTO9 is a nucleic acid stain used for fluorescent labeling of DNA and RNA in living cells. It binds to both double-stranded and single-stranded nucleic acids, emitting green fluorescence upon binding.
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Sypro ruby biofilm matrix stain by Thermo Fisher Scientific
Sourced in United States
SYPRO Ruby Biofilm Matrix Stain is a fluorescent stain used to detect and quantify extracellular matrix components in biofilms. The stain binds to a variety of extracellular polysaccharides, proteins, and other macromolecules.
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Filmtracer sypro ruby by Zeiss
Sourced in Germany
The FilmTracer SYPRO Ruby is a lab equipment product designed for the detection and quantification of proteins in gel electrophoresis. It utilizes the SYPRO Ruby staining method to label proteins, allowing for their visualization under ultraviolet light.
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Filmtracer fm 1 43 by Thermo Fisher Scientific
Sourced in United Kingdom, Canada
FilmTracer FM 1-43 is a lipophilic styryl dye used to visualize biological membranes. It exhibits fluorescence enhancement upon binding to lipid bilayers.

More about "Extracellular Polymeric Substance Matrix"

Extracellular Polymeric Substance (EPS) Matrix: A Complex, Hydrated Network of Microbial Secretions.
This EPS matrix, also known as the extracellular matrix (ECM), is a crucial component of biofilms, providing structural integrity, facilitating intercellular communication, and playing a pivotal role in microbial ecology, bioremediation, and biomedicine.
The EPS matrix is composed of a diverse array of macromolecules, including polysaccharides, proteins, nucleic acids, and other biomolecules.
These components work together to form a hydrated, gel-like structure that encases and protects microbial communities.
The EPS matrix acts as a scaffold, allowing cells to adhere to surfaces and to each other, while also serving as a medium for the exchange of nutrients, signaling molecules, and waste products.
Techniques such as FilmTracer SYPRO Ruby Biofilm Matrix Stain, SYTO 9 Green Fluorescent Nucleic Acid Stain, and Ruthenium red can be used to visualize and analyze the EPS matrix.
Additionally, Alexa Fluor 647-labeled dextran conjugate and Proteinase K can be employed to probe the matrix's permeability and composition, respectively.
SYTO9 and SYPRO Ruby Biofilm Matrix Stain are commonly used to stain and quantify the EPS matrix and biofilm formation.
Understanding the structure, function, and dynamics of the EPS matrix is crucial for advancing research in areas such as microbial ecology, bioremediation, and biomedicine.
By leveraging the insights provided by the EPS matrix, researchers can develop innovative strategies for controlling biofilm formation, enhancing bioremediation processes, and exploring new therapeutic approaches.