Browsing by Subject "S. Aureus"
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Staphylococcus aureus taudinaiheuttajana ja yhteisviljelmämenetelmän käyttö antimikrobiseulonnassa (2010)Staphylococcus aureus is a common commensal and significant opportunistic pathogen. It causes a wide range of infections from superficial skin infections to serious invasive infections. Its pathogenicity is affected by many factors, such as different surface proteins as well as the excretion of toxins and extracellular enzymes. It has many ways to defend a host defense system, such as the formation of capsule and small-colony variants as well as intracellular hiding. Treatment of infections is hindered due to its ability to form resistance to almost every antimicrobial agent used. So far the development of a working and effective vaccine has not been successful. The discovery of new antibacterial agents seems to be still the only efficient way to fight against resistant bacterial strains. However, the development of new antibacterial agents has proved to be difficult. Developing new screening methods is important in order for new drugs to reach the market more effectively and to ensure that new derivatives are more effective and safer. The experimental part of this study aimed at establishing a co-culture of host cells and a pathogen, and to investigate active compounds from primary screen with the established method (Kleymann and Werling 2004). Host cells in the co-culture was HL (Human Lung) cell line and the pathogen was S. aureus (ATCC 25923). Experimental work began by determining bacterial colony-forming units (CFU) and its correlation with absorbance. Based on CFU-determinations the bacterial concentration in the culture media was calculated. Next, the method was optimized and validated. In optimization, statistical parameters S/B-, S/N-values, and Z'-factor were used. Method was optimized regarding cell and bacterial concentrations and incubation time. The method was validated using known antimicrobials. Screening of compounds to be studied was carried out in two stages. All the compounds were first screened in a primary screen. The primary screening method was a standard antibacterial measurement based on turbidometry. Those compounds that were active in the primary screen were investigated in a secondary screen with a co-culture method, but none of the studied compounds showed antimicrobial activity against S. aureus. Therefore we studied the impact of medium that was used in the co-culture method to the activity of the compounds. It was found that the medium had a significant effect on the antibacterial activity of the compounds, the activity was weakened in the presence of the medium. In conclusion, w the established co-culture method is a powerful way to obtain simultaneously information on antibacterial activity as well as cytotoxicity, and it is well suited for further testing of promising compounds.
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(2015)Periodontitis is a globally significant disease which destroys the attachment tissues and alveolar bone of teeth, eventually leading to tooth loss. Biofilms, the most intrinsic lifestyle of bacteria, play a pivotal role in the occurrence of this disease. Periodontal biofilms can be treated with topically administered chlorhexidine and strain-specific antibiotics. However, these antimicrobials do not offer solutions for periodontal attachment tissue and alveolar bone loss. Some therapeutical alternatives for these conventional treatments have been investigated. In numerous studies, periodontitis is treated successfully (increased attachment and/or alveolar bone levels) with topically and systemically administered bisphosphonates. Furthermore, a topically administered bone graft substitute (bioactive glass) has shown to improve periodontal parameters. In addition, bioactive glass has known antimicrobial and anti-biofilm effects. Moreover, a few bisphosphonates have shown antimicrobial activity against some bacterial strains. Hence, both bisphosphonates and bioactive glass are promising materials for dental applications, also raising interest in their combination. Indeed, it could be hypothesized that this combination product could simultaneously treat both the underlying cause (biofilms) and consequences (alveolar bone and attachment tissue loss) of periodontitis. Open research questions remain for the combination product. Is the anti-biofilm effect enhanced when bioactive glass is combined with bisphosphonates? Moreover, do bisphosphonates have intrinsic anti-biofilm properties? These questions are investigated in this thesis, which is a continuation of a recent doctoral dissertation. In this dissertation, a clodronate-bioactive glass combination product was studied by applying it into periodontal pockets. However, anti-biofilm effects were not assessed. In this thesis, a close examination is carried out on these effects, utilizing relevant biofilm models. The aims of this work were to investigate anti-biofilm effects of bisphosphonates (alendronate, clodronate, etidronate, risedronate and zoledronate) (i) alone, administered as solutions and (ii) combined with bioactive glass S53P4. Optimization of the used assay methods (96-well plate assay, Static Biofilm method) was performed. The anti-biofilm effects of bisphosphonate solutions were screened in the 96-well plate assay using a model organism Staphylococcus aureus Newman and a periodontopathogen Aggregatibacter actinomycetemcomitans ATCC 33384. After this, experiments were conducted with bisphosphonate-bioactive glass combinations. The experiments were performed with a single-specie (A. actinomycetemcomitans ATCC 33384) dental biofilm model based on the Static Biofilm method. The model mimics conditions encountered by periodontal bacteria in the oral cavity. In this part, bisphosphonate particle sizes were measured to determine a suitable control material. In addition to bacterial experiments, pH measurements were carried out to gain an insight to a possible anti-biofilm mechanism. Bisphosphonates administered as stand-alone compounds did not have an effect on either the Gram-positive model organism (S. aureus Newman) or the Gram-negative periodontopathogen (A. actinomycetemcomitans ATCC 33384). In contrast, most combinations of bisphosphonate-bioactive glass revealed a statistically significant increase in anti-biofilm effect on A. actinomycetemcomitans ATCC 33384. The combinations were compared to a control composed of inert glass and bioactive glass. In these assay conditions, the risedronate-bioactive glass-combination was the most effective (significant statistical difference, p < 0.05). Other combinations also reduced biofilms (significant statistical differences, p < 0.05), with the exception of clodronate-bioactive glass, where the change was not statistically significant. The most effective combinations (containing risedronate and etidronate) subjected the biofilms to a period of low pH. Conversely, the least effective combination (clodronate-bioactive glass) rapidly became alkaline, similarly to the control compounds (inert glass and bioactive glass). Thus, anti-biofilm efficacy could be connected to lowered pH. This observation is supported by recent literature where A. actinomycetemcomitans has been deemed highly sensitive to acidity. However, establishing the anti-biofilm rank order of bisphosphonate-bioactive glass combinations would benefit from experiments with equal bisphosphonate particle sizes.
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