Publicação
New therapeutic strategies against Staphylococcus epidermidis biofilms
| Resumo: | Staphylococcus epidermidis was previously regarded as an innocuous commensal microorganism on the human skin. However, nowadays it is seen as an important opportunistic pathogen and ranks first among the causative agents of nosocomial infections on indwelling medical devices. Infections with this leading pathogen are characterized by biofilm development on devices (heart valves, catheters, contact lenses, etc.) and this factor is considered the main virulence mechanism of S. epidermidis. Biofilm cells are organised into structured communities enclosed within a matrix of extracellular material. These cells are phenotypically different from planktonic or suspended cells; notably, they resist host defences and display a significantly decreased susceptibility to antimicrobial agents. Since biofilm-associated infections are frequently resistant to conventional antimicrobial therapy, the aims of this doctoral work were to study new therapeutic strategies for treatment of infections caused by S. epidermidis. To this end, the susceptibility of planktonic and biofilm cells to farnesol, a possible antimicrobial agent against S. epidermidis, as well as the effect of farnesol on structure and composition of biofilm matrix were studied. The comparison of the effect of this compound with antibiotics traditionally used in the treatment of S. epidermidis infections such as vancomycin, tetracycline and rifampicin, and the determination of the presence of synergy of farnesol when combined with the antibiotics previously mentioned and with N-acetylcysteine (NAC) were other goals of the work. Moreover, the postantimicrobial effect (PAE) of farnesol and of the antibiotics mentioned above were determined and compared. Antibiotics combination represents a therapeutic option in the treatment of Staphylococcus infections, as a result of the increasing appearance of multi-resistant microorganisms. Taking this into consideration, this work also aimed at studying the effect of a wide range of antibiotics alone and in combination. Finally, the last purpose of the described work was the study of the genetic expression of some genes responsible for virulence of S. epidermidis biofilm cells, icaA (virulence gene - PNAG producing) and rsbU (stress regulator), after being exposed to various treatment conditions. The results showed that farnesol caused a significant reduction of cellular viability of planktonic cells and a less pronounced effect was observed on biofilm cells. The quantification of extracellular polymers and the visualization of biofilms treated with farnesol under confocal microscopy, support the hypothesis that farnesol causes disruption of the cytoplasmic membrane and consequently release of cellular content. Additionally to cell death, farnesol seems also to destroy the biofilm structure and the biofilm matrix reducing the amount of poly-Nacetylglucosamine (PNAG) exopolysaccharide in the biofilm matrix. This general impairment of the biofilm caused by farnesol may be a potential help to the human immune system to eradicate focus of Staphylococcus epidermidis infections. With few exceptions, none of the antibiotics tested and NAC worked in synergy with farnesol. In some cases, farnesol was as effective as the antibiotics tested, being a possible alternative to antibiotics. Furthermore, farnesol has demonstrated to have a pronounced PAE comparatively to the antibiotics tested. All our results suggest farnesol as a potential antimicrobial therapeutic agent against S. epidermidis infections. Another potential alternative to antibiotics may be the use of NAC as a therapeutic agent, since it had a pronounced antimicrobial effect on both planktonic cells and biofilms. To overcome the problem of resistance to antibiotics, we also tested the susceptibility of biofilm cells to double combinations of antibiotics, and some combinations demonstrated to be effective against S. epidermidis biofilms, namely those containing rifampicin. Two of these combinations were rifampicin+clindamycin and rifampicin+gentamicin. These two combinations induced a lower genetic expression of icaA and rsbU genes, responsible for PNAG/PIA production and consequently can reduce biofilm formation recidivism, in comparison with rifampicin alone. This seems to be an additional advantage of the combinatorial therapy over monotherapy. |
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| Autores principais: | Gomes, F. I. |
| Ano: | 2010 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | português |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Staphylococcus epidermidis was previously regarded as an innocuous commensal microorganism on the human skin. However, nowadays it is seen as an important opportunistic pathogen and ranks first among the causative agents of nosocomial infections on indwelling medical devices. Infections with this leading pathogen are characterized by biofilm development on devices (heart valves, catheters, contact lenses, etc.) and this factor is considered the main virulence mechanism of S. epidermidis. Biofilm cells are organised into structured communities enclosed within a matrix of extracellular material. These cells are phenotypically different from planktonic or suspended cells; notably, they resist host defences and display a significantly decreased susceptibility to antimicrobial agents. Since biofilm-associated infections are frequently resistant to conventional antimicrobial therapy, the aims of this doctoral work were to study new therapeutic strategies for treatment of infections caused by S. epidermidis. To this end, the susceptibility of planktonic and biofilm cells to farnesol, a possible antimicrobial agent against S. epidermidis, as well as the effect of farnesol on structure and composition of biofilm matrix were studied. The comparison of the effect of this compound with antibiotics traditionally used in the treatment of S. epidermidis infections such as vancomycin, tetracycline and rifampicin, and the determination of the presence of synergy of farnesol when combined with the antibiotics previously mentioned and with N-acetylcysteine (NAC) were other goals of the work. Moreover, the postantimicrobial effect (PAE) of farnesol and of the antibiotics mentioned above were determined and compared. Antibiotics combination represents a therapeutic option in the treatment of Staphylococcus infections, as a result of the increasing appearance of multi-resistant microorganisms. Taking this into consideration, this work also aimed at studying the effect of a wide range of antibiotics alone and in combination. Finally, the last purpose of the described work was the study of the genetic expression of some genes responsible for virulence of S. epidermidis biofilm cells, icaA (virulence gene - PNAG producing) and rsbU (stress regulator), after being exposed to various treatment conditions. The results showed that farnesol caused a significant reduction of cellular viability of planktonic cells and a less pronounced effect was observed on biofilm cells. The quantification of extracellular polymers and the visualization of biofilms treated with farnesol under confocal microscopy, support the hypothesis that farnesol causes disruption of the cytoplasmic membrane and consequently release of cellular content. Additionally to cell death, farnesol seems also to destroy the biofilm structure and the biofilm matrix reducing the amount of poly-Nacetylglucosamine (PNAG) exopolysaccharide in the biofilm matrix. This general impairment of the biofilm caused by farnesol may be a potential help to the human immune system to eradicate focus of Staphylococcus epidermidis infections. With few exceptions, none of the antibiotics tested and NAC worked in synergy with farnesol. In some cases, farnesol was as effective as the antibiotics tested, being a possible alternative to antibiotics. Furthermore, farnesol has demonstrated to have a pronounced PAE comparatively to the antibiotics tested. All our results suggest farnesol as a potential antimicrobial therapeutic agent against S. epidermidis infections. Another potential alternative to antibiotics may be the use of NAC as a therapeutic agent, since it had a pronounced antimicrobial effect on both planktonic cells and biofilms. To overcome the problem of resistance to antibiotics, we also tested the susceptibility of biofilm cells to double combinations of antibiotics, and some combinations demonstrated to be effective against S. epidermidis biofilms, namely those containing rifampicin. Two of these combinations were rifampicin+clindamycin and rifampicin+gentamicin. These two combinations induced a lower genetic expression of icaA and rsbU genes, responsible for PNAG/PIA production and consequently can reduce biofilm formation recidivism, in comparison with rifampicin alone. This seems to be an additional advantage of the combinatorial therapy over monotherapy. |
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