Dental caries is a biofilm-related disease, widely perceived to be caused by oral ecological imbalance when cariogenic/aciduric bacteria obtain an ecological advantage. Compared with planktonic bacteria, dental plaques are difficult to remove under extracellular polymeric substance protection. In this study, the effect of caffeic acid phenethyl ester (CAPE) on a preformed cariogenic multi-species biofilm was evaluated, which was comprised of cariogenic bacteria (Streptococcus mutans), commensal bacteria (Streptococcus gordonii), and a pioneer colonizer (Actinomyces naeslundii). Our result revealed that treatment with 0.08 mg/mL CAPE reduced live S. mutans in the preformed multi-species biofilm while not significantly changing the quantification of live S. gordonii. CAPE significantly reduced the production of lactic acid, extracellular polysaccharide, and extracellular DNA and made the biofilm looser. Moreover, CAPE could promote the H2O2 production of S. gordonii and inhibit the expression of SMU.150 encoding mutacin to modulate the interaction among species in biofilms. Overall, our results suggested that CAPE could inhibit the cariogenic properties and change the microbial composition of the multi-species biofilms, indicating its application potential in dental caries prevention and management.
Microorganisms embedded within an extracellular polymeric matrix are known as biofilm. The extensive use of antibiotics to overcome the biofilm-linked challenges has led to the emergence of multidrug-resistant strains. Staphylococcus aureus is one such nosocomial pathogen that is known to cause biofilm-linked infections. Thus, novel strategies have been adopted in this study to inhibit the biofilm formation of S. aureus. Two natural compounds, namely, 1,4-naphthoquinone (a quinone derivative) and tryptophan (aromatic amino acid), have been chosen as they could independently show efficient antibiofilm activity. To enhance the antibiofilm potential, the two compounds were combined and tested against the same organism. Several experiments like crystal violet (CV) assay, protein estimation, extracellular polymeric substance (EPS) extraction, and estimation of metabolic activity confirmed that the combination of the two compounds could significantly inhibit the biofilm formation of S. aureus. To comprehend the underlying mechanism, efforts were further directed to understand whether the two compounds could inhibit biofilm formation by compromising the cell surface hydrophobicity of the bacteria. The results revealed that the cell surface hydrophobicity got reduced by ~ 49% when the compounds were applied together. Thus, the combinations could show enhanced antibiofilm activity by attenuating cell surface hydrophobicity. Further studies revealed that the selected concentrations of the compounds could disintegrate (~ 70%) the pre-existing biofilm of the test bacteria without showing any antimicrobial activity. Hence, the combined application of tryptophan and 1,4-naphthoquinone could be used to inhibit the biofilm threats of S. aureus.
- MeSH
- antibakteriální látky farmakologie MeSH
- biofilmy MeSH
- lidé MeSH
- matrix extracelulárních polymerních látek MeSH
- mikrobiální testy citlivosti MeSH
- stafylokokové infekce * farmakoterapie mikrobiologie MeSH
- Staphylococcus aureus * MeSH
- tryptofan farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Introduction.Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) are the most common pathogens from the genus Staphylococcus causing biofilm-associated infections. Generally, biofilm-associated infections represent a clinical challenge. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections.Gap Statement. A variety of protocols for in vitro drug activity testing against staphylococcal biofilms have been introduced. However, there are often fundamental differences. All these differences in methodical approaches can then be reflected in the form of discrepancies between results.Aim. In this study, we aimed to develop optimal conditions for staphylococcal biofilm formation on pegs. The impact of peg surface modification was also studied.Methodology. The impact of tryptic soy broth alone or supplemented with foetal bovine serum (FBS) or human plasma (HP), together with the impact of the inoculum density of bacterial suspensions and the shaking versus the static mode of cultivation, on total biofilm biomass production in SA and SE reference strains was studied. The surface of pegs was modified with FBS, HP, or poly-l-lysine (PLL). The impact on total biofilm biomass was evaluated using the crystal violet staining method and statistical data analysis.Results. Tryptic soy broth supplemented with HP together with the shaking mode led to crucial potentiation of biofilm formation on pegs in SA strains. The SE strain did not produce biofilm biomass under the same conditions on pegs. Preconditioning of peg surfaces with FBS and HP led to a statistically significant increase in biofilm biomass formation in the SE strain.Conclusion. Optimal cultivation conditions for robust staphylococcal biofilm formation in vitro might differ among different bacterial strains and methodical approaches. The shaking mode and supplementation of cultivation medium with HP was beneficial for biofilm formation on pegs for SA (ATCC 29213) and methicillin-resistant SA (ATCC 43300). Peg conditioning with HP and PLL had no impact on biofilm formation in either of these strains. Peg coating with FBS showed an adverse effect on the biofilm formation of these strains. By contrast, there was a statistically significant increase in biofilm biomass production on pegs coated with FBS and HP for SE (ATCC 35983).
- MeSH
- bakteriologické techniky přístrojové vybavení metody MeSH
- biofilmy klasifikace účinky léků růst a vývoj MeSH
- biomasa MeSH
- druhová specificita MeSH
- kultivační média chemie farmakologie MeSH
- lidé MeSH
- matrix extracelulárních polymerních látek klasifikace účinky léků MeSH
- Staphylococcus klasifikace účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
This study is focused on the analysis of extracellular DNA (eDNA) from a biofilm matrix formed by Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica. The presence of eDNA in the biofilm of all the studied strains was confirmed by confocal laser scanning microscopy using fluorescent dyes with high affinity to nucleic acid. The protocol for eDNA isolation from the biofilm matrix was established, and subsequent characterization of the eDNA was performed. The purified eDNA obtained from the biofilm matrix of all three microorganisms was compared to the genomic DNA (gDNA) isolated from relevant planktonic grown cells. The process of eDNA isolation consisted of biofilm cultivation, its collection, sonication, membrane filtration, dialysis, lyophilisation, and extraction of DNA separated from the biofilm matrix with cetyltrimethylammonium bromide. An amplified fragment length polymorphism (AFLP) was used for comparing eDNA and gDNA. AFLP profiles showed a significant similarity between eDNA and gDNA at the strain level. The highest similarity, with a profile concordance rate of 94.7% per strain, was observed for S. aureus, L. monocytogenes, and S. enterica exhibited lower profiles similarity (78% and 60%, respectively). The obtained results support the hypothesis that the eDNA of studied bacterial species has its origin in the gDNA.
Alogenní transplantace pankreatu je jediná možnost terapie diabetu, která vede k dlouhodobé normoglykemii bez rizika hypoglykemie. Vzhledem k nedostatku dárců orgánů a nežádoucím účinkům imunosuprese je transplantační léčba v diabetologii vyhrazena pouze vybraným pacientům a základem terapie diabetu nadále zůstává exogenní aplikace inzulinu. S nedostatkem orgánů k transplantaci se ovšem potýkají i ostatní medicínské obory, ve kterých je transplantační léčba život zachraňujícím výkonem při terminálním selhání orgánů. Ve snaze obejít nedostatek dárcovských orgánů jsou rozvíjeny nové léčebné metody postavené na základech buněčné terapie a tkáňového inženýrství. K přípravě tzv. bioarteficiálních orgánů jsou hojně využívány komponenty extracelulární matrix, připravené metodou decelularizace orgánů a tkání. Vytvořená bezbuněčná nativní proteinová lešení jsou znovu osídlena vybranými druhy buněk. Proteiny extracelulární matrix buňkám zprostředkovávají signály k migraci, diferenciaci, proliferaci a podporují jejich funkce. V laboratorních podmínkách se takto podařilo vytvořit funkční tkáně a jednoduché orgány, které jsou již aplikovatelné in vivo. Komplexní orgány mají před sebou ještě optimalizaci příprav. Metoda se však jeví nadějně, a to i pro tzv. bioarteficiální pankreas, na jehož přípravách pracuje náš tým Laboratoře Langerhansových ostrůvků IKEM.
Allogeneic pancreas transplantation as diabetes mellitus treatment option results in long-term normoglycemia without hypoglycemia risk. In diabetology, transplantation therapy is assigned only to selected patients. Regarding the shortage of organ donors and adverse effects of immunosuppressive drugs, exogenous insulin therapy remains the standard. New methods based on cell therapy and tissue engineering are being developed to evade the donor organ shortage. Extracellular matrix components prepared by organ and tissue decellularization are used for development of „bioartificial“ organs. Acellular native protein scaffolds are then recellularized by selected cell types. Extracellular matrix proteins mediate cell migration, differentiation, proliferation and promote cell functions. Functional tissues and simple organs have been prepared using this method in vitro, which are now applicable in vivo. Preparation of complex organs is yet to be optimized. However, the methodology seems promising for the „bioartificial“ pancreas, which is being prepared by our team in the Laboratory of Pancreatic Islets in IKEM
- MeSH
- diabetes mellitus 1. typu terapie MeSH
- extracelulární matrix * MeSH
- homologní transplantace MeSH
- imunosupresivní léčba MeSH
- lidé MeSH
- matrix extracelulárních polymerních látek MeSH
- transplantace kmenových buněk MeSH
- transplantace Langerhansových ostrůvků metody MeSH
- transplantace slinivky břišní * metody MeSH
- Check Tag
- lidé MeSH
Contrary to the planktonic state of bacteria, their biofilm form represents severe complications in areas such as human medicine or food industry due to the increasing resistance against harsh conditions and treatment. In the present study, infrared attenuated total reflection (IR-ATR) spectroscopy has been applied as an analytic tool studying Escherichia coli ( E. coli) biofilm formation close to real time. We report on IR spectroscopic investigations on the biofilm formation via ATR waveguides probing the biofilm in the spectral window of 1800-900 cm-1 at dynamic flow conditions, which facilitated monitoring the growth dynamics during several days. Key IR bands are in the range 1700-1590 cm-1 (amide I), 1580-1490 cm-1 (amide II), and 1141-1006 cm-1 extracellular polymeric substances (EPS), which were evaluated as a function of time. Cyclic fluctuations of the amide I and amide II bands and a continuous increase of the EPS band were related to the starvation of bottom-layered bacteria caused by the nutrient gradient. Potential death of bacteria may then result in cannibalistic behavior known for E. coli colonies. Observing this behavior via IR spectroscopy allows revealing these cyclical changes in bottom-layered bacteria within the biofilm under continuous nutrient flow, in molecular detail, and during extended periods for the first time.
Odstranění mikrobiálního biofilmu a potlačení jeho tvorby jsou v současné době velkou výzvou, která vyžaduje citlivější a účinnější strategie s ohledem k nárůstu mikrobiálních rezistencí. Nanočástice zaměřené na eradikaci biofilmu vyvolávají v posledních deseti letech zájem kvůli svým vlastnostem. Tyto nanočástice mají široké spektrum biologických účinků a mezi nimiž je významná antibiofilmová aktivita. Daří se jim proniknout do biofilmové matrice, která působí jako bariéra pro mnoho antibiotik a dezinfekčních prostředků.
Eradication of microbial biofilm and prevention of its formation is currently a major challenge that requires more sensitive and effective strategies due to increasing resistence of microorganisms. Nanoparticles targeted on biofilm eradication have gained enormous popularity over the last ten years due to their unique properties. These nanoparticles have a wide range of biological applications, including a significant antibiofilm activity. They are able to penetrate into a biofilm matrix that serves as a barrier against many antibiotics and disinfectants.
- MeSH
- antiinfekční látky farmakologie izolace a purifikace MeSH
- biofilmy účinky léků MeSH
- hořčík MeSH
- matrix extracelulárních polymerních látek mikrobiologie účinky léků MeSH
- nanočástice * chemie terapeutické užití MeSH
- oxid zinečnatý MeSH
- stříbro MeSH
- železo MeSH
- zlato MeSH
- Publikační typ
- práce podpořená grantem MeSH
