A brief exploration of EPS composition in biofilms of Staphylococcus spp ATCC reference strains

2020 ◽  
Author(s):  
Cristina D. Cruz ◽  
Rebekah C. Hewitt ◽  
Päivi Tammela
Keyword(s):  
Biofilm Formation ◽  
Extracellular Polymeric Substances ◽  
Bacterial Biofilm ◽  
Biofilm Growth ◽  
Time Points ◽  
Microtiter Plates ◽  
The Matrix ◽  
Sulfuric Acid Method ◽  
Staphylococcus Spp ◽  
Physical And Chemical

<p>Antibiotic resistance is expected to cause 10 million deaths per year worldwide by 2050. One of the mechanisms for the resilient nature of bacteria toward antibiotics is through the formation of biofilm. Bacterial biofilms are sessile communities of microorganisms, which exist in a matrix of proteins, carbohydrates, eDNA and other various components – collectively known as extracellular polymeric substances. Biofilms slow the penetration of drugs, and also contribute to the development of a resistant phenotype known as persisters. Thus, understanding biofilm composition might contribute to the development of anti-biofilm strategies. The aim of this study was to explore biofilm formed by five <em>Staphylococcus</em> spp ATCC strains, commonly used in research as references: <em>S. aureus</em> 25923, <em>S. aureus</em> 29213, <em>S. aureus</em> 43300 (methicillin-resistant), <em>S. aureus</em> 6538 and <em>S. epidermidis</em> 12228. Biofilm mass and its components were analysed after 24h and 72h of biofilm growth. Bacterial biofilm was prepared in 96-well microtiter plates, in Trypticase Soy Broth supplemented with 1% glucose. After incubation at 37°C, absorbance measurements and crystal violet staining were performed and the specific biofilm formation determined for each strain. Extracellular polymeric substances were extracted using a combination of physical and chemical methods; including centrifugation, vortexing and the use of 1.5M NaCl. In these assays, biofilms were grown in polystyrene tubes containing 10 ml of same media mentioned above. The concentration of protein, carbohydrate and eDNA was determined using the Bicinchoninic acid assay, phenol-sulfuric acid method and DNeasy<sup>®</sup> Blood and Tissue Kit, respectively, followed by spectroscopy. Our data demonstrated heterogeneity between the biofilm-forming capabilities and EPS components within staphylococcal strains and species. Strains 25923 and 6538 had the highest value for biofilm formation at both time points. Interestingly, strain 43300 was the only one to show a significant increase in biofilm after 72h. Contradictory to previous findings, <em>S. epidermidis</em> 12228 was found to be a good biofilm producer. At both time points studied, strains demonstrated considerably higher concentrations of protein (varying from 172 µg/mL – 345 µg/mL) and carbohydrate (56 µg/mL - 372µg/mL) in EPS compared to eDNA (2.74 µg/mL – 8.12 µg/mL). On average, strains 43300 and 12228 had the highest concentration of protein, and the latter also had the highest carbohydrate and eDNa amounts at 72h. Strains 25923 and 6538 had a significant decrease in eDNA concentration over time. Based on this brief study, the relative quantities of EPS components investigated is similar to that of other studies with protein being the most plentiful component followed by carbohydrate and then considerably lower amounts of eDNA. Differences in specific biofilm formation did not directly reflect variations observed in abundance of a particular constituent in the matrix of EPS. This study also showed that <em>S. epidermidis</em> 12228, usually classified as a weak or non-biofilm former, was able to grow a relatively substantial biofilm under the conditions tested here.</p>

Antibiotics Application Strategies to Control Biofilm Formation in Pathogenic Bacteria

2020 ◽  
Vol 21 (4) ◽  
pp. 270-286 ◽  
Author(s):  
Fazlurrahman Khan ◽  
Dung T.N. Pham ◽  
Sandra F. Oloketuyi ◽  
Young-Mog Kim
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Extracellular Polymeric Substances ◽  
Quorum Quenching ◽  
Resistance Mechanisms ◽  
Bacterial Biofilm ◽  
Bacterial Cells ◽  
High Concentration ◽  
Biofilm Inhibition ◽  
Abiotic Surfaces

Background: The establishment of a biofilm by most pathogenic bacteria has been known as one of the resistance mechanisms against antibiotics. A biofilm is a structural component where the bacterial community adheres to the biotic or abiotic surfaces by the help of Extracellular Polymeric Substances (EPS) produced by bacterial cells. The biofilm matrix possesses the ability to resist several adverse environmental factors, including the effect of antibiotics. Therefore, the resistance of bacterial biofilm-forming cells could be increased up to 1000 times than the planktonic cells, hence requiring a significantly high concentration of antibiotics for treatment. Methods: Up to the present, several methodologies employing antibiotics as an anti-biofilm, antivirulence or quorum quenching agent have been developed for biofilm inhibition and eradication of a pre-formed mature biofilm. Results: Among the anti-biofilm strategies being tested, the sub-minimal inhibitory concentration of several antibiotics either alone or in combination has been shown to inhibit biofilm formation and down-regulate the production of virulence factors. The combinatorial strategies include (1) combination of multiple antibiotics, (2) combination of antibiotics with non-antibiotic agents and (3) loading of antibiotics onto a carrier. Conclusion: The present review paper describes the role of several antibiotics as biofilm inhibitors and also the alternative strategies adopted for applications in eradicating and inhibiting the formation of biofilm by pathogenic bacteria.

Archaeal biofilms: widespread and complex

2013 ◽  
Vol 41 (1) ◽  
pp. 393-398 ◽  
Author(s):  
Sabrina Fröls
Keyword(s):  
Biofilm Formation ◽  
Extracellular Polymeric Substances ◽  
Bacterial Species ◽  
Surface Adhesion ◽  
Form Complex ◽  
Abiotic Surfaces ◽  
Archaeal Species ◽  
Physical And Chemical ◽  
Insight Into ◽  
Mixed Communities

Biofilms or multicellular structures become accepted as the dominant microbial lifestyle in Nature, but in the past they were only studied extensively in bacteria. Investigations on archaeal monospecies cultures have shown that many archaeal species are able to adhere on biotic and abiotic surfaces and form complex biofilm structures. Biofilm-forming archaea were identified in a broad range of extreme and moderate environments. Natural biofilms observed are mostly mixed communities composed of archaeal and bacterial species of various abundances. The physiological functions of the archaea identified in such mixed communities suggest a significant impact on the biochemical cycles maintaining the flow and recycling of the nutrients on earth. Therefore it is of high interest to investigate the characteristics and mechanisms underlying the archaeal biofilm formation. In the present review, I summarize and discuss the present investigations of biofilm-forming archaeal species, i.e. their diverse biofilm architectures in monospecies or mixed communities, the identified EPSs (extracellular polymeric substances), archaeal structures mediating surface adhesion or cell–cell connections, and the response to physical and chemical stressors implying that archaeal biofilm formation is an adaptive reaction to changing environmental conditions. A first insight into the molecular differentiation of cells within archaeal biofilms is given.

Effect of Human Milk and its Components on Streptococcus Mutans Biofilm Formation

2015 ◽  
Vol 39 (3) ◽  
pp. 255-261 ◽  
Author(s):  
LM Allison ◽  
LA Walker ◽  
BJ Sanders ◽  
Z Yang ◽  
G Eckert ◽  
...  
Keyword(s):  
Breast Milk ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Current Knowledge ◽  
Post Partum ◽  
Human Breast Milk ◽  
Human Breast ◽  
Flat Bottom ◽  
Biofilm Growth ◽  
Microtiter Plates

Objective: This study investigated the effects of human breast milk and its components on the nutritional aspect of the caries process due to Streptococcus mutans UA159 biofilm formation. Study design: Human breast milk was collected from 11 mothers during 3-9 months postpartum. To test for the effect on biofilm formation, a 16-hour culture of S. mutans was treated with dilutions of human breast milk and several major components of human breast milk, lactose, lactoferrin, IgA, and bovine casein in sterile 96-well flat bottom microtiter plates for 24 hours. The biofilms were fixed, washed, stained with crystal violet, and extracted. Absorbance was measured to evaluate biofilm growth mass. Results: Dilutions 1:10-1:2,560 of the human breast milk samples increased biofilm formation by 1.5-3.8 fold compared to the control. Lactoferrin decreased biofilm formation significantly in all dilutions (average milk concentration of 3 mg/ml). Lactose had no effect at average breast milk concentrations (60 mg/ml) except at its lowest concentration (15 mg/ml) where it was increased. IgA significantly decreased biofilm formation at its highest concentration of 2,400 μg/ml (average milk concentration 600 μg/ml). Casein caused significantly increased biofilm formation at all concentrations tested above the average milk content (2.3 mg/ml). Conclusions: The results of this study demonstrate an increase in S. mutans biofilm formation by human breast milk 3-9 months post partum. Among its major components, only casein significantly increased biofilm formation among the concentrations analyzed. Lactose had no effect except at 15 mg/ml. Lactoferrin and IgA significantly decreased S. mutans biofilm formation at their highest concentrations. This information expands the current knowledge regarding the nutritional influence of breastfeeding and validates the necessity to begin an oral hygiene regimen once the first tooth erupts.

scholarly journals Bacterial Biofilm Growth on 3D-Printed Materials

2021 ◽  
Vol 12 ◽  
Author(s):  
Donald C. Hall ◽  
Phillip Palmer ◽  
Hai-Feng Ji ◽  
Garth D. Ehrlich ◽  
Jarosław E. Król
Keyword(s):  
Medical Devices ◽  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Bacterial Attachment ◽  
Bacterial Biofilm ◽  
Chronic Infections ◽  
Biofilm Growth ◽  
Wide Range ◽  
3D Printed ◽  
Printed Materials

Recent advances in 3D printing have led to a rise in the use of 3D printed materials in prosthetics and external medical devices. These devices, while inexpensive, have not been adequately studied for their ability to resist biofouling and biofilm buildup. Bacterial biofilms are a major cause of biofouling in the medical field and, therefore, hospital-acquired, and medical device infections. These surface-attached bacteria are highly recalcitrant to conventional antimicrobial agents and result in chronic infections. During the COVID-19 pandemic, the U.S. Food and Drug Administration and medical officials have considered 3D printed medical devices as alternatives to conventional devices, due to manufacturing shortages. This abundant use of 3D printed devices in the medical fields warrants studies to assess the ability of different microorganisms to attach and colonize to such surfaces. In this study, we describe methods to determine bacterial biofouling and biofilm formation on 3D printed materials. We explored the biofilm-forming ability of multiple opportunistic pathogens commonly found on the human body including Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus to colonize eight commonly used polylactic acid (PLA) polymers. Biofilm quantification, surface topography, digital optical microscopy, and 3D projections were employed to better understand the bacterial attachment to 3D printed surfaces. We found that biofilm formation depends on surface structure, hydrophobicity, and that there was a wide range of antimicrobial properties among the tested polymers. We compared our tested materials with commercially available antimicrobial PLA polymers.

The phycobiota-derived proteins Pµ84 and Pµ19 suppress bacterial biofilm formation in gram-negative pathogens

2020 ◽  
Author(s):  
Yuchen Han ◽  
Wolfgang R. Streit ◽  
Ines Krohn
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Bacterial Biofilm ◽  
Klebsiella Pneumonia ◽  
Biofilm Inhibition ◽  
Marine Systems ◽  
Microtiter Plates ◽  
Small Proteins ◽  
Ongoing Work ◽  
Growth Promoting

<p>Microalgae are typically found in freshwater and marine systems and they harbor a mostly a beneficial growth promoting microbiota. We have recently isolated several small proteins from the microbiomes of microalga (<em>Scenedesmus quadricauda</em>, <em>Microasterias crux-melintensis</em>, <em>Chlorella saccherophilia</em>) and have tested them for their role in either inhibition of biofilm formation and/or biofilm degradation. Thereby we have identified two candidate proteins that showed promising activities on biofilm inhibition and degradation. These proteins were designated Pµ84 and Pµ19 and strongly affected biofilm formation in several human- and plant-pathogenic bacteria. Recombinant and purified Pµ84 and Pµ19 were applied in biofilm assays in microtiter plates and reduced biofilms formed by <em>Stenotrophomonas maltophilia</em>, <em>Pseudomonas aeruginosa</em> and <em>Klebsiella pneumonia</em>. If expressed in the different hosts, biofilms were reduced by a factor of 40% and if applied as exogenous proteins, biofilms were reduced up to 20%. Pµ84 application also resulted in a delayed biofilm formation and biofilm formation was affected by a factor of 17%. The microprotein Pµ19 consist of 57 aa and Pµ84 consists of 49 aa. Ongoing work elucidates the mechanism of Pµ84 and Pµ19 on the reduction of biofilm in order to achieve the optimal activity.</p>

scholarly journals Immobilizing hydrolytic active Papain on biodegradable PLLA for biofilm inhibition in cardiovascular applications

2020 ◽  
Vol 6 (3) ◽  
pp. 172-175
Author(s):  
Michael Teske ◽  
Tina Kießlich ◽  
Niels Grabow ◽  
Sabine Illner ◽  
Julia Fischer ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Hydrolytic Enzyme ◽  
Bacterial Biofilm ◽  
Biofilm Inhibition ◽  
Biofilm Growth ◽  
Clostridioides Difficile ◽  
Adhesive Surface

AbstractThe use of biomaterials in medicine is becoming increasingly important. One of the main concerns is the foreign body associated infection caused by direct microbial contamination or clinical infections. The bacterial biofilm formation on biomaterials depends on their surface properties. Therefore, several anti-adhesive surface modifications were developed. Nevertheless, the demand for antimicrobial agents that prevent bacterial colonisation is still largely unmet. The immobilization of active antimicrobial agents, such as antibacterial peptides or enzymes, offers a potential approach to achieve long-lasting effectiveness. In this investigation, the hydrolytic enzyme papain with its published antibacterial activity was covalently immobilized on the well-established biodegradable biomaterial poly-L-lactic acid (PLLA). For the characterization of the enzymes on the PLLA surfaces, the protein content and enzyme activity were determined. A biofilm assay was performed to test the effect of the papain-modified PLLA samples on the biofilm-forming bacterial strain Clostridioides difficile, one of the most frequently occurring human nosocomial pathogens. The investigated hydrolytic enzyme papain could be immobilized by coupling via the crosslinker EDC to the PLLA surface. Detection was performed by determination of the amount of protein and the reduced biofilm growth after 24 h and 72 h compared to the reference.

scholarly journals Effects ofBacillusSerine Proteases on the Bacterial Biofilms

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Olga Mitrofanova ◽  
Ayslu Mardanova ◽  
Vladimir Evtugyn ◽  
Lydia Bogomolnaya ◽  
Margarita Sharipova
Keyword(s):  
Biofilm Formation ◽  
Quantitative Methods ◽  
Time Course ◽  
Extracellular Polymeric Substances ◽  
Proteolytic Enzymes ◽  
Opportunistic Pathogen ◽  
Glutamyl Endopeptidase ◽  
Bacterial Cells ◽  
Biofilm Growth ◽  
Tract Infections

Serratia marcescensis an emerging opportunistic pathogen responsible for many hospital-acquired infections including catheter-associated bacteremia and urinary tract and respiratory tract infections. Biofilm formation is one of the mechanisms employed byS. marcescensto increase its virulence and pathogenicity. Here, we have investigated the main steps of the biofilm formation byS. marcescensSR 41-8000. It was found that the biofilm growth is stimulated by the nutrient-rich environment. The time-course experiments showed thatS. marcescenscells adhere to the surface of the catheter and start to produce extracellular polymeric substances (EPS) within the first 2 days of growth. After 7 days,S. marcescensbiofilms maturate and consist of bacterial cells embedded in a self-produced matrix of hydrated EPS. In this study, the effect ofBacillus pumilus3-19 proteolytic enzymes on the structure of 7-day-oldS. marcescensbiofilms was examined. Using quantitative methods and scanning electron microscopy for the detection of biofilm, we demonstrated a high efficacy of subtilisin-like protease and glutamyl endopeptidase in biofilm removal. Enzymatic treatment resulted in the degradation of the EPS components and significant eradication of the biofilms.

scholarly journals Metabolic Remodeling during Biofilm Development ofBacillus subtilis

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Tippapha Pisithkul ◽  
Jeremy W. Schroeder ◽  
Edna A. Trujillo ◽  
Ponlkrit Yeesin ◽  
David M. Stevenson ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Regulatory Networks ◽  
Developmental Process ◽  
Central Carbon Metabolism ◽  
Bacterial Biofilm ◽  
Biofilm Growth ◽  
Time Resolved ◽  
Content Type ◽  
Metabolic Remodeling ◽  
Biofilm Development

ABSTRACTBiofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such asBacillus subtilishas been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development inB. subtilisby combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring duringB. subtilisbiofilm development that will serve as a useful road map for future studies on biofilm physiology.IMPORTANCEBacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such asBacillus subtilis, very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development inB. subtilisby combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.

Effects of Cola-Flavored Beverages and Caffeine on Streptococcus mutans Biofilm Formation and Metabolic Activity

2017 ◽  
Vol 41 (4) ◽  
pp. 294-299 ◽  
Author(s):  
Roger P Dotsey ◽  
Elizabeth A S Moser ◽  
George J Eckert ◽  
Richard L Gregory
Keyword(s):  
Streptococcus Mutans ◽  
Metabolic Activity ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Bacterial Biofilm ◽  
Water Soluble ◽  
High Fructose Corn Syrup ◽  
Biofilm Growth ◽  
High Fructose

Objective: To examine the effects of cola-flavored beverages and caffeine on growth and metabolism of Streptococcus mutans biofilm. This study was designed to determine if carbonated beverages or caffeine can increase S. mutans growth and biofilm formation and metabolic activity in vitro, potentially leading to increased S. mutans-associated cariogenicity in children that consume them. Study Design: Six different cola-flavored products, plus pure caffeine, and pure high fructose corn syrup (HFCS), at different concentrations similar to those in the beverages were tested. A 16-hour culture of S. mutans was treated with different dilutions in bacteriological media. To test for the effect on biofilm formation, the biofilm was stained with crystal violet. The absorbance was determined to evaluate biofilm growth. Biofilm metabolic activity was measured based on biofilm having the ability to reduce XTT to a water-soluble orange compound. Results: The inclusion of HFCS in the beverages, as well as pure HFCS, significantly enhanced bacterial biofilm formation and metabolic activity. Pure caffeine and the presence of caffeine in beverages did not significantly increase biofilm formation, but pure caffeine significantly increased metabolism, and Diet Coke had significantly greater metabolic activity than Caffeine-Free Diet Coke. Conclusions: HFCS increases both the biofilm formation and metabolism of S. mutans, and caffeine in some cases increases metabolism of S. mutans.

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