Abstract
Interspecies interactions are vital for the development of any complex communities including multispecies biofilms, which are receiving increasing attention due to their ubiquitous presence in most natural but also man-made habitats. Several studies have shown that species residing within complex bacterial communities interact both inter and intra-specifically, and that these interactions are instrumental in shaping the community structure and distribution of bacterial species within multispecies biofilms. These complex interactions often lead to emergent properties in biofilms, such as enhanced tolerance against antibiotics, host immune responses, and other stresses, which have been shown to provide benefits to the biofilm members. Co-culturing studies using in-vitro multispecies settings have shown these to enhance the overall biomass produced and many studies have revealed the formation of microbial aggregates, microcolonies or biofilm formation as a response to grazing. Predation by bacteriovorous protists can influence physiological status of the bacterial communities and can result in bacterial responses at the community and species levels, which is in turn influenced by the interplay of several complex interactions and parameters.
In the introductory sections, a comprehensive overview is provided of our current understanding of multispecies biofilms, biofilm development and interactions between FLP and bacteria in these biofilms. Special attention is given to predator-prey interactions.
This doctoral thesis aims to address various aspects of bacterial interactions inducing multispecies biofilm formation and the role of biofilms to serve as a protective growth environment under grazing pressure. More specifically, we examined microbial composition and diversity of multispecies biofilms associated with toothbrushes and dishwashers, including FLP occurrence on toothbrushes, and characterized a range of bacterial communities in dishwasher systems with respect to the species ability to form biofilms individually and in co-cultures. Further, the influence of bacterial interactions and its impact on population dynamics in a four-species bacterial model system under grazing pressure was investigated.
This PhD thesis has resulted in 3 published manuscripts in peer-reviewed journals and one draft manuscript. The manuscripts follow the order of my work on describing the inter-bacterial interactions with a focus on biofilm formation and its protective effects observed for different bacterial species under grazing pressure.
In Manuscript 1, special attention was paid to the occurrence of FLP on toothbrushes. In total, 6 out of 28 toothbrushes were FLP positive. We show that FLP and bacteria, including some opportunistic pathogens, were detected and identified from toothbrushes. Amoebae were the dominant FLP morphotype recovered from toothbrush samples, which may be due to the fact that amoebae have a higher attachment capacity compared to other FLP morphotypes. Bacterial isolates identified in this study which are classified as opportunistic pathogens include Acinetobacter johnsonii, Enterobacter faecalis, Enterobacter cloacae, Klebsiella oxytoca, Staphylococcus aureus and Streptococcus salivarius. Toothbrush head design had a significant influence on bacterial diversity and composition where designed heads fitted with additional projections had a reduced bacterial load on their surfaces compared to conventional toothbrushes. The result from this study corroborates previous findings that closely arranged bristles on toothbrushes increase microbial retention.
Biofilm associated microbial communities can thrive in extreme or hostile environments, where growing as individuals members could be challenging. This aspect was investigated in manuscripts 2 and 3. In Manuscript 2, microbial composition of a man-made system that is household dishwashers that offer challenging conditions for microbial survival was determined using next generation sequencing. Growth limiting factors like high temperatures in the rnage between 30 – 80 °C, varying pH levels ranging 7-12, high salt concentrations, use of detergents and mechanical shear generated from water ejectors during washing cycles constrain microbial survival in this extreme system. 24 different household dishwashers were investigated for both fungal and bacterial diversity within the biofilm formed on the rubber seals. In most samples, bacterial genera such as Pseudomonas, Escherichia and Acinetobacter, known to include opportunistic pathogens, were represented. The most frequently encountered fungal genera in these samples belonged to Candida, Cryptococcus and Rhodotorula and representatives of Candida spp. were found at highest prevalence in all sampled dishwashers. It was also observed that conditions of dishwashers including its age, usage frequency, and the hardness of the influent water supply to these dishwashers had a significant impact on bacterial and fungal composition. Pairwise correlations revealed certain bacterial groups to co-occur and so did the fungal groups. Early adhesion, contact and interactions were speculated to be vital in the process of mixed fungal- bacterial biofilm, where complexes of the two, bacteria and fungi, could provide a preliminary biogenic structure for biofilm establishment. In Manuscript 3, four dishwashers were selected and screened the composition of bacteria and fungi, isolated from a defined area of one square centimeter of rubber from 4 domestic dishwashers. A total of 80 isolates (64 bacterial and 16 fungal) were obtained and tested for their ability to form multispecies biofilms in-vitro. 32 out of 140 tested (23%) four-species bacterial combinations displayed consistent synergism leading to an overall increase in biomass. Bacterial isolates from two of the four dishwashers generated a high fraction of synergistically interacting four-species consortia. Furthermore, two synergistic four-species bacterial consortia were tested for their ability to incorporate an opportunistic fungal pathogen, Exophiala dermatitidis, and establish as biofilms on sterile ethylene propylene diene monomer M-class (EPDM) rubber and polypropylene (PP) surfaces. When the bacterial consortia included E. dermatitidis, the overall cell numbers of both bacteria and fungi increased and a substantial increase in biofilm biomass was observed. This study indicates a novel phenomenon of cross-kingdom synergy in biofilm formation and further studies are needed to determine their
potential implications for human health. Our research shows that persisting poly-extremotolerant groups of microorganisms in household appliances are well established under these unfavorable conditions, supported by the biofilm mode of growth.
Bacteria protect itself from a predator through various mechanisms and one such mechanism is biofilm formation that has been shown to confer protection against grazing. While in nature, as most biofilms were known to harbor different bacterial species, less is known on the effect of grazing with respect to multispecies biofilm settings. Towards this aspect, in Manuscript 4, the effects of grazing on the interactions and dynamics of a multispecies bacterial consortium by a pelagic protozoan predator were investigated. Mono- and multispecies biofilms of four bacterial soil isolates, namely Xanthomonas retroflexus, Stenotrophomonas rhizophila, Microbacterium oxydans and Paenibacillus amylolyticus, were cultivated and subjected to grazing by the ciliate Tetrahymena pyriformis. Grazing strongly reduced the planktonic cell numbers of P. amylolyticus, S. rhizophila and X. retroflexus in monocultures while the cell numbers in the underlying biofilms of S. rhizophila and X. retroflexus increased, but not in P. amylolyticus. This may be due to the fact that while grazing enhanced biofilm formation in the former two species, no biofilm was formed by P. amylolyticus in monoculture, either with or without grazing. However, in four-species biofilms, biofilm formation was observed to be higher than in the best monoculture and a strong biodiversity effect was observed in the presence of grazing. While cell numbers of X. retroflexus, S. rhizophila, and P. amylolyticus in the planktonic fraction were greatly reduced in the presence of grazers, cell numbers of all three species strongly increased in the multispecies biofilm. Our results demonstrate that synergistic interactions between the four-species induced biofilm formation and further suggest that the best biofilm producer X. retroflexus when exposed to the grazer not only protect itself but also extend supported to other members that were sensitive to grazing, indicating a scenario of “shared grazing protection” within the four-species biofilm model.
In conclusion, this PhD thesis demonstrates that studies using multispecies conditions, though low in complexity comapred to natural bacterial communities, still enable us to get closer to natural settings where biofilm communities are often present as multispecies microbial communities. The resulting emergent functions, like increased biomass production and fitness benefits (e.g. grazer protection) associated within biofilm architecture, underline the prevalence of synergistic interactions in multispecies biofilms and their impact on individual species during biofilm development.
In the introductory sections, a comprehensive overview is provided of our current understanding of multispecies biofilms, biofilm development and interactions between FLP and bacteria in these biofilms. Special attention is given to predator-prey interactions.
This doctoral thesis aims to address various aspects of bacterial interactions inducing multispecies biofilm formation and the role of biofilms to serve as a protective growth environment under grazing pressure. More specifically, we examined microbial composition and diversity of multispecies biofilms associated with toothbrushes and dishwashers, including FLP occurrence on toothbrushes, and characterized a range of bacterial communities in dishwasher systems with respect to the species ability to form biofilms individually and in co-cultures. Further, the influence of bacterial interactions and its impact on population dynamics in a four-species bacterial model system under grazing pressure was investigated.
This PhD thesis has resulted in 3 published manuscripts in peer-reviewed journals and one draft manuscript. The manuscripts follow the order of my work on describing the inter-bacterial interactions with a focus on biofilm formation and its protective effects observed for different bacterial species under grazing pressure.
In Manuscript 1, special attention was paid to the occurrence of FLP on toothbrushes. In total, 6 out of 28 toothbrushes were FLP positive. We show that FLP and bacteria, including some opportunistic pathogens, were detected and identified from toothbrushes. Amoebae were the dominant FLP morphotype recovered from toothbrush samples, which may be due to the fact that amoebae have a higher attachment capacity compared to other FLP morphotypes. Bacterial isolates identified in this study which are classified as opportunistic pathogens include Acinetobacter johnsonii, Enterobacter faecalis, Enterobacter cloacae, Klebsiella oxytoca, Staphylococcus aureus and Streptococcus salivarius. Toothbrush head design had a significant influence on bacterial diversity and composition where designed heads fitted with additional projections had a reduced bacterial load on their surfaces compared to conventional toothbrushes. The result from this study corroborates previous findings that closely arranged bristles on toothbrushes increase microbial retention.
Biofilm associated microbial communities can thrive in extreme or hostile environments, where growing as individuals members could be challenging. This aspect was investigated in manuscripts 2 and 3. In Manuscript 2, microbial composition of a man-made system that is household dishwashers that offer challenging conditions for microbial survival was determined using next generation sequencing. Growth limiting factors like high temperatures in the rnage between 30 – 80 °C, varying pH levels ranging 7-12, high salt concentrations, use of detergents and mechanical shear generated from water ejectors during washing cycles constrain microbial survival in this extreme system. 24 different household dishwashers were investigated for both fungal and bacterial diversity within the biofilm formed on the rubber seals. In most samples, bacterial genera such as Pseudomonas, Escherichia and Acinetobacter, known to include opportunistic pathogens, were represented. The most frequently encountered fungal genera in these samples belonged to Candida, Cryptococcus and Rhodotorula and representatives of Candida spp. were found at highest prevalence in all sampled dishwashers. It was also observed that conditions of dishwashers including its age, usage frequency, and the hardness of the influent water supply to these dishwashers had a significant impact on bacterial and fungal composition. Pairwise correlations revealed certain bacterial groups to co-occur and so did the fungal groups. Early adhesion, contact and interactions were speculated to be vital in the process of mixed fungal- bacterial biofilm, where complexes of the two, bacteria and fungi, could provide a preliminary biogenic structure for biofilm establishment. In Manuscript 3, four dishwashers were selected and screened the composition of bacteria and fungi, isolated from a defined area of one square centimeter of rubber from 4 domestic dishwashers. A total of 80 isolates (64 bacterial and 16 fungal) were obtained and tested for their ability to form multispecies biofilms in-vitro. 32 out of 140 tested (23%) four-species bacterial combinations displayed consistent synergism leading to an overall increase in biomass. Bacterial isolates from two of the four dishwashers generated a high fraction of synergistically interacting four-species consortia. Furthermore, two synergistic four-species bacterial consortia were tested for their ability to incorporate an opportunistic fungal pathogen, Exophiala dermatitidis, and establish as biofilms on sterile ethylene propylene diene monomer M-class (EPDM) rubber and polypropylene (PP) surfaces. When the bacterial consortia included E. dermatitidis, the overall cell numbers of both bacteria and fungi increased and a substantial increase in biofilm biomass was observed. This study indicates a novel phenomenon of cross-kingdom synergy in biofilm formation and further studies are needed to determine their
potential implications for human health. Our research shows that persisting poly-extremotolerant groups of microorganisms in household appliances are well established under these unfavorable conditions, supported by the biofilm mode of growth.
Bacteria protect itself from a predator through various mechanisms and one such mechanism is biofilm formation that has been shown to confer protection against grazing. While in nature, as most biofilms were known to harbor different bacterial species, less is known on the effect of grazing with respect to multispecies biofilm settings. Towards this aspect, in Manuscript 4, the effects of grazing on the interactions and dynamics of a multispecies bacterial consortium by a pelagic protozoan predator were investigated. Mono- and multispecies biofilms of four bacterial soil isolates, namely Xanthomonas retroflexus, Stenotrophomonas rhizophila, Microbacterium oxydans and Paenibacillus amylolyticus, were cultivated and subjected to grazing by the ciliate Tetrahymena pyriformis. Grazing strongly reduced the planktonic cell numbers of P. amylolyticus, S. rhizophila and X. retroflexus in monocultures while the cell numbers in the underlying biofilms of S. rhizophila and X. retroflexus increased, but not in P. amylolyticus. This may be due to the fact that while grazing enhanced biofilm formation in the former two species, no biofilm was formed by P. amylolyticus in monoculture, either with or without grazing. However, in four-species biofilms, biofilm formation was observed to be higher than in the best monoculture and a strong biodiversity effect was observed in the presence of grazing. While cell numbers of X. retroflexus, S. rhizophila, and P. amylolyticus in the planktonic fraction were greatly reduced in the presence of grazers, cell numbers of all three species strongly increased in the multispecies biofilm. Our results demonstrate that synergistic interactions between the four-species induced biofilm formation and further suggest that the best biofilm producer X. retroflexus when exposed to the grazer not only protect itself but also extend supported to other members that were sensitive to grazing, indicating a scenario of “shared grazing protection” within the four-species biofilm model.
In conclusion, this PhD thesis demonstrates that studies using multispecies conditions, though low in complexity comapred to natural bacterial communities, still enable us to get closer to natural settings where biofilm communities are often present as multispecies microbial communities. The resulting emergent functions, like increased biomass production and fitness benefits (e.g. grazer protection) associated within biofilm architecture, underline the prevalence of synergistic interactions in multispecies biofilms and their impact on individual species during biofilm development.
| Original language | English |
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| Publisher | Department of Biology, Faculty of Science, University of Copenhagen |
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| Publication status | Published - 2018 |
