Publicação
Influence of oxygen conditions on bacterial interactions within biofilms related with cystic fibrosis
| Resumo: | Cystic Fibrosis (CF) is a genetic disorder associated with multispecies infections where interactions between classical and newly identified bacteria might be crucial for a better understanding of their persistent colonization in CF lungs. Nonetheless, little is known about the contributions of these microbes in the development of chronic biofilms, particularly under variable oxygen environments that are known to occur in vivo in the airways of CF patients. As such, this work aimed at giving insights into the physiology, phenotype and ecology of polymicrobial communities involving traditional (Staphylococcus aureus and Pseudomonas aeruginosa) and emergent bacteria (Achromobacter baumannii, Dolosigranulum pigrum, Inquilinus limosus, Klebsiella pneumoniae and Stenotrophomonas maltophilia) associated to CF. At a first stage of this work, the ability of abovementioned bacteria to growth planktonically and to develop biofilms under in vitro atmospheres with different oxygen concentrations (aerobic and anaerobic) was examined. Results showed that all bacteria were able to growth and to develop biofilms under such conditions, demonstrating high number of cultivable cells even with a significant decreasing in the amount of biomass for low-oxygen atmospheres. Based on these initial findings, that revealed an easy adaptation of the emergent-species to the CF airways environments, it was considered crucial to investigate how they interact and contribute to the polymicrobial consortia when cultured with CF-common pathogens. As such, S. aureus, I. limosus and S. maltophilia were grown in dual-species populations with P. aeruginosa under variable oxygen atmospheres and these biofilms were thoroughly characterized for biomass, colony-forming units (CFU) and relative distribution of bacterial populations. Results demonstrated that dual-species biofilms, similarly to most single-species biofilms, produced more biomass under aerobic conditions. However, the presence of S. aureus, I. limosus and S. maltophilia in co-culture with P. aeruginosa significantly reduced the biofilm biomass formed comparatively with the mono-species P. aeruginosa biofilm, although the number of cultivable cells was not affected. Regarding microbial composition, the results obtained by CFU counting and PNA FISH under aerobic and anaerobic atmospheres, demonstrated that in all polymicrobial consortia, P. aeruginosa was still the dominant species. The latest results have shown that populations encompassing CF-bacteria could easily adapt to planktonic and biofilm state under variable oxygen conditions resembling CF. But how these microorganisms contribute to disease progression and to antibiotic therapy was still to be unveiled. As such, those bacterial populations were grown under variable oxygen conditions and their antibiotic resistance profiles using ciprofloxacin were assessed. Results indicate that biofilms were notoriously more difficult to eradicate than their planktonic counterparts, for all oxygen atmospheres. Regarding polymicrobial populations, biofilm eradication was not achieved by using monotherapy, showing even an increased overall cell density when compared with mono-species P. aeruginosa biofilm, in all oxygen conditions. In general, biofilm compositions changed as a result of antibiotic treatment, with alterations being dependent on the antibiotic concentration and oxygen conditions implemented. For consortia formed between P. aeruginosa and S. aureus, the latter species predominated in the consortia for both oxygen conditions. Contrariwise, the consortia encompassing P. aeruginosa – I. limosus and P. aeruginosa – S. maltophilia were dominated by the CF-key pathogen P. aeruginosa. As such, the endurance of P. aeruginosa within the consortia, before and after antibiotic treatment, could be the basis for a higher contribution of this species to the antibiotic resistance presented by dual-species biofilms. However, the increasing survival of S. maltophilia and I. limosus (slight increase for I. limosus) in dual-species consortia with P. aeruginosa after antibiotic exposure, for all oxygen atmospheres, indicates that these species may have also a preponderant role in increasing the whole resistance within the consortia. The PNA FISH method was employed to directly localize and discriminate the bacterial populations within the consortia, corroborating the dominance of P. aeruginosa within the mixed-species consortia (determined by CFU counting), and allowed to observe a decreasing in the overall cell density for all consortia under low-oxygen atmospheres. In summary, the results demonstrated that emergent- and traditional-species are able to live in association with key-CF pathogen P. aeruginosa commonly found in CF airways under variable oxygen atmospheres, developing highly resilient consortia towards antibiotic treatment. The interactions established between emergent-species and other major pathogens might be crucial to understanding the persistent microbial infection in CF airways and bring information about the pathogenic character of such emergent species. |
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| Autores principais: | Magalhães, Andreia Patrícia Alves |
| Assunto: | Engenharia e Tecnologia::Biotecnologia Industrial |
| Ano: | 2015 |
| 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: | Cystic Fibrosis (CF) is a genetic disorder associated with multispecies infections where interactions between classical and newly identified bacteria might be crucial for a better understanding of their persistent colonization in CF lungs. Nonetheless, little is known about the contributions of these microbes in the development of chronic biofilms, particularly under variable oxygen environments that are known to occur in vivo in the airways of CF patients. As such, this work aimed at giving insights into the physiology, phenotype and ecology of polymicrobial communities involving traditional (Staphylococcus aureus and Pseudomonas aeruginosa) and emergent bacteria (Achromobacter baumannii, Dolosigranulum pigrum, Inquilinus limosus, Klebsiella pneumoniae and Stenotrophomonas maltophilia) associated to CF. At a first stage of this work, the ability of abovementioned bacteria to growth planktonically and to develop biofilms under in vitro atmospheres with different oxygen concentrations (aerobic and anaerobic) was examined. Results showed that all bacteria were able to growth and to develop biofilms under such conditions, demonstrating high number of cultivable cells even with a significant decreasing in the amount of biomass for low-oxygen atmospheres. Based on these initial findings, that revealed an easy adaptation of the emergent-species to the CF airways environments, it was considered crucial to investigate how they interact and contribute to the polymicrobial consortia when cultured with CF-common pathogens. As such, S. aureus, I. limosus and S. maltophilia were grown in dual-species populations with P. aeruginosa under variable oxygen atmospheres and these biofilms were thoroughly characterized for biomass, colony-forming units (CFU) and relative distribution of bacterial populations. Results demonstrated that dual-species biofilms, similarly to most single-species biofilms, produced more biomass under aerobic conditions. However, the presence of S. aureus, I. limosus and S. maltophilia in co-culture with P. aeruginosa significantly reduced the biofilm biomass formed comparatively with the mono-species P. aeruginosa biofilm, although the number of cultivable cells was not affected. Regarding microbial composition, the results obtained by CFU counting and PNA FISH under aerobic and anaerobic atmospheres, demonstrated that in all polymicrobial consortia, P. aeruginosa was still the dominant species. The latest results have shown that populations encompassing CF-bacteria could easily adapt to planktonic and biofilm state under variable oxygen conditions resembling CF. But how these microorganisms contribute to disease progression and to antibiotic therapy was still to be unveiled. As such, those bacterial populations were grown under variable oxygen conditions and their antibiotic resistance profiles using ciprofloxacin were assessed. Results indicate that biofilms were notoriously more difficult to eradicate than their planktonic counterparts, for all oxygen atmospheres. Regarding polymicrobial populations, biofilm eradication was not achieved by using monotherapy, showing even an increased overall cell density when compared with mono-species P. aeruginosa biofilm, in all oxygen conditions. In general, biofilm compositions changed as a result of antibiotic treatment, with alterations being dependent on the antibiotic concentration and oxygen conditions implemented. For consortia formed between P. aeruginosa and S. aureus, the latter species predominated in the consortia for both oxygen conditions. Contrariwise, the consortia encompassing P. aeruginosa – I. limosus and P. aeruginosa – S. maltophilia were dominated by the CF-key pathogen P. aeruginosa. As such, the endurance of P. aeruginosa within the consortia, before and after antibiotic treatment, could be the basis for a higher contribution of this species to the antibiotic resistance presented by dual-species biofilms. However, the increasing survival of S. maltophilia and I. limosus (slight increase for I. limosus) in dual-species consortia with P. aeruginosa after antibiotic exposure, for all oxygen atmospheres, indicates that these species may have also a preponderant role in increasing the whole resistance within the consortia. The PNA FISH method was employed to directly localize and discriminate the bacterial populations within the consortia, corroborating the dominance of P. aeruginosa within the mixed-species consortia (determined by CFU counting), and allowed to observe a decreasing in the overall cell density for all consortia under low-oxygen atmospheres. In summary, the results demonstrated that emergent- and traditional-species are able to live in association with key-CF pathogen P. aeruginosa commonly found in CF airways under variable oxygen atmospheres, developing highly resilient consortia towards antibiotic treatment. The interactions established between emergent-species and other major pathogens might be crucial to understanding the persistent microbial infection in CF airways and bring information about the pathogenic character of such emergent species. |
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