Publicação
Insights into the ecology of polymicrobial biofilms involved in cystic fibrosis
| Resumo: | Cystic Fibrosis (CF), a lethal hereditary disorder, is characterized by high rates of morbidity and mortality caused by pulmonary microbial infections. Pseudomonas aeruginosa is typically the prevailing pathogen in the airways of CF patients, but advances in molecular technologies have disclosed an emergent and diverse microbial community inhabiting CF lungs. For most of these microbes, the pathogenesis, clinical relevance, adaptation to CF airways niche, antimicrobial susceptibilities and even interactions with other CF-associated pathogens remains poorly understood. As such, this work aimed at giving insights into the physiology, phenotype and ecology of polymicrobial communities involving traditional and emergent bacteria associated to CF. Therefore, Inquilinus limosus and Dolosigranulum pigrum, two emergent bacteria that have been frequently recovered from CF airways, as well as the CF-classical pathogen P. aeruginosa, were used throughout this work. At a first stage of this work, the ability of I. limosus and D. pigrum to develop single- and dual-species biofilms together with P. aeruginosa under standard aerobic conditions and to resist towards a range of antibiotics from varied classes was examined. Results showed that both emergent bacteria produced significantly less biomass than P. aeruginosa and displayed greater sensitivity to antibiotics when singlespecies biofilms were formed. However, when grown in dual-species consortia with P. aeruginosa, the presence of I. limosus and D. pigrum was crucial in increasing the antibiotic-resistance of the overall consortia, independently of the reduced biofilm biomass formed and a decrease in biofilm matrix content. Based on these initial findings that revealed microbial interactions between the CF-emergent species with a CF-major pathogen and knowing that zones with steep oxygen gradients occur within the CF airways, it was considered pivotal to investigate the biofilm-forming ability of such individual species and their antibiotic susceptibilities under oxygen-variable atmospheres (aerobic, microaerophilic and anaerobic). Results demonstrated that all bacteria were able to develop monospecies biofilms under different atmospheres. Unlike earlier results, the emergent biofilms displayed multi-drug resistance under aerobic conditions, which was higher than for P. aeruginosa, and endured even for low-oxygen environments. Such findings reveal that I. limosus, D. pigrum and P. aeruginosa seem to easily adapt to the CF airways environments. Fluorescence in situ hybridization using peptide nucleic acid probes (PNA FISH) has been pinpointed as a valuable molecular tool for microbial identification and discrimination within polymicrobial communities. As such, two PNA probes were designed and developed to accurately distinguish and localize P. aeruginosa and I. limosus within the polymicrobial consortia. These PNA probes - Paer565 and Ilim569 - yielded high theoretical specificities (~100% for both) and sensitivities (90.3 and 87.5%, respectively for Paer565 and Ilim569) in identifying the corresponding target microorganisms (P. aeruginosa and I. limosus). Posteriorly, a multiplex PNA FISH assay combined with 4',6-diamidino-2-phenylindole, DAPI, (to identify D. pigrum) was validated for the precise discrimination of the three populations within mixed-species biofilms. Previous findings have shown that I. limosus and D. pigrum could easily adapt to the CF airways environments, but how they interacted and contributed to the polymicrobial consortia with CF-common pathogens, under such conditions, was still to be disclosed. As such, I. limosus and D. pigrum were grown in dual- and three-species populations with P. aeruginosa under variable oxygen atmospheres and the biofilms were thoroughly characterized for biomass, respiratory activity, colony-forming units (CFU) number, antibiotic resistance profiles, and relative distributions of bacterial populations. The PNA FISH method developed before was employed to directly localize and discriminate the bacterial populations within the consortia. Results showed that multi-species biofilms displayed enhanced antibiotic-resistance compared with single-species biofilms. Dual-species biofilms displayed the lowest CFU reductions after antibiotic treatment and even less under low-oxygen atmospheres. Regarding microbial composition, dual-species biofilms presented similar bacterial proportions, whereas P. aeruginosa and D. pigrum dominated the threespecies consortia, with I. limosus as the smallest representative population. In general, biofilm compositions changed in result of antibiotic treatment, with alterations being dependent on the antibiotic, concentration and oxygen conditions used. The consortia encompassing I. limosus and P. aeruginosa were dominated by the latter species in aerobiosis. However, the survival of I. limosus towards antibiotics under anaerobic conditions led this species to occupy a significant portion together with P. aeruginosa in the overall biofilm, which indicates that I. limosus had a preponderant role in increasing the whole resistance of biofilms, particularly under anaerobiosis. The three-species biofilms displayed the highest sensitivity (the eradication was achieved for levofloxacin and ciprofloxacin), with D. pigrum and/or P. aeruginosa dominating and I. limosus declining for most cases. It is concluded, therefore, that the preponderance of D. pigrum in the biofilm was decisive to decrease I. limosus and lead to an increase in overall sensitivity of the biofilm to a large number of antibiotics. PNA FISH allowed the direct observation of the location and distribution of the three-species species within the biofilms, corroborating the dominance of D. pigrum and P. aeruginosa within the mixed-species consortia (determined by CFU counting) and facilitating the understanding of the real complex interactions among the bacterial species. In summary, I. limosus and D. pigrum could easily adapt and survive as biofilms, a common trait associated to virulence, under oxygen conditions resembling the airways of CF patients. Both species were also able to grow associated with the common pathogen P. aeruginosa, leading to polymicrobial consortia with antibiotic resilience and shaping social interactions in the consortia. Interestingly, this work offers a new approach in antibiotherapy targeting biofilm-related infections based on disturbing one member of the community to facilitate the subsequent treatment of the biofilm by antibiotics. Hence, these studies should be extended to other emergent microbes and encourage rethinking the in-use CF antibiotic treatment and eventually the search for new safe and efficient antimicrobial strategies targeting biofilms that may cause severe infection, thus ensuring quality of care and minimizing the risk of mortality in CF patients. |
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| Autores principais: | Lopes, Susana Patrícia |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Cystic Fibrosis (CF), a lethal hereditary disorder, is characterized by high rates of morbidity and mortality caused by pulmonary microbial infections. Pseudomonas aeruginosa is typically the prevailing pathogen in the airways of CF patients, but advances in molecular technologies have disclosed an emergent and diverse microbial community inhabiting CF lungs. For most of these microbes, the pathogenesis, clinical relevance, adaptation to CF airways niche, antimicrobial susceptibilities and even interactions with other CF-associated pathogens remains poorly understood. As such, this work aimed at giving insights into the physiology, phenotype and ecology of polymicrobial communities involving traditional and emergent bacteria associated to CF. Therefore, Inquilinus limosus and Dolosigranulum pigrum, two emergent bacteria that have been frequently recovered from CF airways, as well as the CF-classical pathogen P. aeruginosa, were used throughout this work. At a first stage of this work, the ability of I. limosus and D. pigrum to develop single- and dual-species biofilms together with P. aeruginosa under standard aerobic conditions and to resist towards a range of antibiotics from varied classes was examined. Results showed that both emergent bacteria produced significantly less biomass than P. aeruginosa and displayed greater sensitivity to antibiotics when singlespecies biofilms were formed. However, when grown in dual-species consortia with P. aeruginosa, the presence of I. limosus and D. pigrum was crucial in increasing the antibiotic-resistance of the overall consortia, independently of the reduced biofilm biomass formed and a decrease in biofilm matrix content. Based on these initial findings that revealed microbial interactions between the CF-emergent species with a CF-major pathogen and knowing that zones with steep oxygen gradients occur within the CF airways, it was considered pivotal to investigate the biofilm-forming ability of such individual species and their antibiotic susceptibilities under oxygen-variable atmospheres (aerobic, microaerophilic and anaerobic). Results demonstrated that all bacteria were able to develop monospecies biofilms under different atmospheres. Unlike earlier results, the emergent biofilms displayed multi-drug resistance under aerobic conditions, which was higher than for P. aeruginosa, and endured even for low-oxygen environments. Such findings reveal that I. limosus, D. pigrum and P. aeruginosa seem to easily adapt to the CF airways environments. Fluorescence in situ hybridization using peptide nucleic acid probes (PNA FISH) has been pinpointed as a valuable molecular tool for microbial identification and discrimination within polymicrobial communities. As such, two PNA probes were designed and developed to accurately distinguish and localize P. aeruginosa and I. limosus within the polymicrobial consortia. These PNA probes - Paer565 and Ilim569 - yielded high theoretical specificities (~100% for both) and sensitivities (90.3 and 87.5%, respectively for Paer565 and Ilim569) in identifying the corresponding target microorganisms (P. aeruginosa and I. limosus). Posteriorly, a multiplex PNA FISH assay combined with 4',6-diamidino-2-phenylindole, DAPI, (to identify D. pigrum) was validated for the precise discrimination of the three populations within mixed-species biofilms. Previous findings have shown that I. limosus and D. pigrum could easily adapt to the CF airways environments, but how they interacted and contributed to the polymicrobial consortia with CF-common pathogens, under such conditions, was still to be disclosed. As such, I. limosus and D. pigrum were grown in dual- and three-species populations with P. aeruginosa under variable oxygen atmospheres and the biofilms were thoroughly characterized for biomass, respiratory activity, colony-forming units (CFU) number, antibiotic resistance profiles, and relative distributions of bacterial populations. The PNA FISH method developed before was employed to directly localize and discriminate the bacterial populations within the consortia. Results showed that multi-species biofilms displayed enhanced antibiotic-resistance compared with single-species biofilms. Dual-species biofilms displayed the lowest CFU reductions after antibiotic treatment and even less under low-oxygen atmospheres. Regarding microbial composition, dual-species biofilms presented similar bacterial proportions, whereas P. aeruginosa and D. pigrum dominated the threespecies consortia, with I. limosus as the smallest representative population. In general, biofilm compositions changed in result of antibiotic treatment, with alterations being dependent on the antibiotic, concentration and oxygen conditions used. The consortia encompassing I. limosus and P. aeruginosa were dominated by the latter species in aerobiosis. However, the survival of I. limosus towards antibiotics under anaerobic conditions led this species to occupy a significant portion together with P. aeruginosa in the overall biofilm, which indicates that I. limosus had a preponderant role in increasing the whole resistance of biofilms, particularly under anaerobiosis. The three-species biofilms displayed the highest sensitivity (the eradication was achieved for levofloxacin and ciprofloxacin), with D. pigrum and/or P. aeruginosa dominating and I. limosus declining for most cases. It is concluded, therefore, that the preponderance of D. pigrum in the biofilm was decisive to decrease I. limosus and lead to an increase in overall sensitivity of the biofilm to a large number of antibiotics. PNA FISH allowed the direct observation of the location and distribution of the three-species species within the biofilms, corroborating the dominance of D. pigrum and P. aeruginosa within the mixed-species consortia (determined by CFU counting) and facilitating the understanding of the real complex interactions among the bacterial species. In summary, I. limosus and D. pigrum could easily adapt and survive as biofilms, a common trait associated to virulence, under oxygen conditions resembling the airways of CF patients. Both species were also able to grow associated with the common pathogen P. aeruginosa, leading to polymicrobial consortia with antibiotic resilience and shaping social interactions in the consortia. Interestingly, this work offers a new approach in antibiotherapy targeting biofilm-related infections based on disturbing one member of the community to facilitate the subsequent treatment of the biofilm by antibiotics. Hence, these studies should be extended to other emergent microbes and encourage rethinking the in-use CF antibiotic treatment and eventually the search for new safe and efficient antimicrobial strategies targeting biofilms that may cause severe infection, thus ensuring quality of care and minimizing the risk of mortality in CF patients. |
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