Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/99887
Type: Theses
Title: Haemophilus influenzae survival and biofilm formation in a complex physical, chemical and multi-species environment
Author: Tikhomirova, Alexandra
Issue Date: 2016
School/Discipline: School of Biological Sciences
Abstract: H. influenzae is an opportunistic human pathogen capable of occupying a range of niches in the respiratory tract. Both during health and disease processes, H. influenzae must adapt to the conditions present in the microniches it encounters and correspondingly alter its lifestyle in the presence of non-optimal conditions. The niches encountered by H. influenzae in the human host include the presence of chemical stress such as ROS and RCS compounds, as well as a diverse range of pH conditions ranging from pH 7-9, and the presence of other members of the microflora, such as Streptococcus pneumoniae. In this thesis, we have identified that there are strain-specific components related to the adaptation of H. influenzae to specific conditions. We have identified that different H. influenzae isolates employ different mechanisms to adapt to the presence of diverse, and often stress-inducing conditions. One adaptation mechanisms employed by H. influenzae is the adoption of a sessile biofilm lifestyle. We have shown that there is a strain-specific response of H. influenzae isolates in their biofilm formation to the presence of different nutrient conditions, and to the presence of ROS and RCS such as formaldehyde, methylglyoxal and H2O2. We have also shown that in different nutrient conditions, there is a different requirement for eDNA in the EPS matrix of individual strains. In addition, we have identified a role of the nickel import system nikKLMQO-nimR of H. influenzae in its biofilm formation. We have shown that when this system is absent, or when H. influenzae is in a nickel limited environment, the H. influenzae cells display an increased biofilm formation. This biofilm formation response was accompanied by a global transcriptomics response, which displayed global changes in metabolic pathways. Further to these findings, we have shown that there are strain-specific differences in H. influenzae adaptation to different pH conditions. These differences were expressed both as differences in biofilm formation, and differences on the transcriptomics level. Another significant finding was that the pH played an important role in the inter-species interactions of H. influenzae and S. pneumoniae. In a batch culture system in stationary phase at a lower initial pH of 7.4, S. pneumoniae was able to convert H. influenzae into a VBNC state. However, H. influenzae was able to survive in a culturable state in co-culture in log phase, or when a higher initital pH was used. We have also shown that in co-culture, there were significant transcriptomics changes both in H. influenzae and S. pneumoniae, including an induction of stress response genes in H. influenzae, and a down-regulation of sugar utilisation genes in S. pneumoniae. Importantly, we have shown that in a continuous flow cell system, H. influenzae and S. pneumoniae exist in a different lifestyle and different transcriptomics profile than in a batch culture system, both in mono- and co-culture. In this system, the 2 species were able to co-exist without the reduction in culturability in either of the species. We have also shown that the transcriptomic profile in co-culture in a flow cell system is different to what was observed in the batch system, with one of the major findings being the up-regulation of sugar utilisation genes in S. pneumoniae, suggesting the potential metabolic relationship between H. influenzae and S. pneumoniae. We have investigated this finding further, and have indeed demonstrated that nutrient availability and carbon source impact the H. influenzae/ S. pneumoniae interactions. In a flow cell system containing a nutrient-limited CDM media with glucose, H. influenzae was able to survive equally in mono- and co-culture. However, S. pneumoniae was unable to grow in mono-culture, and in co-culture displayed 3 phenotypes: a wild-type phenotype at 24 h, an undetectable state until 336 h, and a small colony variant state at 336 h. S. pneumoniae did not significantly impact the H. influenzae transcriptome in co-culture in either the undetected or SCV state, but did subtly modify the H. influenzae transcriptome upon transition to different time-points of growth, or different nutrient conditions. We have also preliminarily identified transcriptomic changes in S. pneumoniae at 64 h and 336 h, which correspond to a persister-cell like state observed in other bacterial species. Overall, we have identified that environmental factors significantly impact the ability of H. influenzae to survive and to adopt lifestyles pertaining to these environments, in a strain-specific manner. We have shown that these adaptations are often accompanied by global transcriptomics changes. We have also identified that the inter-species interactions between H. influenzae and S. pneumoniae are highly complex and their outcome depends on a multitude of environmental conditions.
Advisor: Kidd, Stephen Peter
Paton, James Cleland
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2016
Keywords: multispecies biofilm
bacterial infection
otitis media
Haemophilus influenzae
Streptococcus pneumoniae
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals
Appears in Collections:Research Theses

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