Publicação
Insights into Pseudomonas aeruginosa and Burkholderia contaminans interactions in cystic fibrosis-associated biofilms infections
| Resumo: | The Gram-negative Pseudomonas aeruginosa and the Burkholderia cepacia complex are opportunistic pathogenic bacteria commonly responsible for severe infections in Cystic Fibrosis (CF) patients. These bacteria form biofilms in the airways, making infections challenging to treat. Recently, some advances have been made to understand the complex interaction between microorganisms and their implications for disease prognosis and therapy response in CF. Therefore, this study aimed to contribute to the knowledge of the potential role of interactions between P. aeruginosa and the emerging B. contaminans during infection, as well as their impact on the action of antibiotics frequently used to control P. aeruginosa. Regarding their biofilm formation ability, the presence of B. contaminans resulted in a decrease in biomass and the number of cultivable cells in dual-species biofilms compared to P. aeruginosa single biofilms after 72h, suggesting an antagonistic interaction between the bacteria. Concerning the role of these microbial interactions in susceptibility to colistin and ciprofloxacin, the Minimum Inhibitory Concentration (MIC) was first determined. For ciprofloxacin, it was determined to be 0.25mg/L for both, while for colistin, the MIC varied between 0.5 and 1mg/L for P. aeruginosa, and it was not possible to determine the MIC of this antibiotic in B. contaminans. In biofilms, P. aeruginosa exhibited higher susceptibility to ciprofloxacin treatment, in the presence of B. contaminans, at concentrations starting at 2 × MIC. Several genes associated with virulence in P. aeruginosa were analyzed, and the specificity of the primer pairs corresponding to the genes pqsE, lasI, and mucA in P. aeruginosa were confirmed by PCR and qPCR. Additionally, RNA extraction was optimized for B. contaminans biofilms, concluding that using 10 biofilms yielded a greater RNA quantity. However, qPCR results indicated that 16S rRNA, the gene chosen for relative expression normalization, was not a suitable reference gene due to significant variation in cycle quantification across different conditions. Thus, further optimizations will be necessary to quantify the expression of the genes of interest. In summary, this study demonstrates how interspecies interactions can influence antibiotic susceptibility, enhancing our understanding of P. aeruginosa and B. contaminans behavior in dual-species biofilms. |
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| Autores principais: | Peixoto, Beatriz da Costa |
| Assunto: | Biofilms P. aeruginosa B. contaminans Interactions Cystic fibrosis Biofilmes Interações Fibrose cística |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | The Gram-negative Pseudomonas aeruginosa and the Burkholderia cepacia complex are opportunistic pathogenic bacteria commonly responsible for severe infections in Cystic Fibrosis (CF) patients. These bacteria form biofilms in the airways, making infections challenging to treat. Recently, some advances have been made to understand the complex interaction between microorganisms and their implications for disease prognosis and therapy response in CF. Therefore, this study aimed to contribute to the knowledge of the potential role of interactions between P. aeruginosa and the emerging B. contaminans during infection, as well as their impact on the action of antibiotics frequently used to control P. aeruginosa. Regarding their biofilm formation ability, the presence of B. contaminans resulted in a decrease in biomass and the number of cultivable cells in dual-species biofilms compared to P. aeruginosa single biofilms after 72h, suggesting an antagonistic interaction between the bacteria. Concerning the role of these microbial interactions in susceptibility to colistin and ciprofloxacin, the Minimum Inhibitory Concentration (MIC) was first determined. For ciprofloxacin, it was determined to be 0.25mg/L for both, while for colistin, the MIC varied between 0.5 and 1mg/L for P. aeruginosa, and it was not possible to determine the MIC of this antibiotic in B. contaminans. In biofilms, P. aeruginosa exhibited higher susceptibility to ciprofloxacin treatment, in the presence of B. contaminans, at concentrations starting at 2 × MIC. Several genes associated with virulence in P. aeruginosa were analyzed, and the specificity of the primer pairs corresponding to the genes pqsE, lasI, and mucA in P. aeruginosa were confirmed by PCR and qPCR. Additionally, RNA extraction was optimized for B. contaminans biofilms, concluding that using 10 biofilms yielded a greater RNA quantity. However, qPCR results indicated that 16S rRNA, the gene chosen for relative expression normalization, was not a suitable reference gene due to significant variation in cycle quantification across different conditions. Thus, further optimizations will be necessary to quantify the expression of the genes of interest. In summary, this study demonstrates how interspecies interactions can influence antibiotic susceptibility, enhancing our understanding of P. aeruginosa and B. contaminans behavior in dual-species biofilms. |
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