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Abstract

Biofilm formation has been recognised as an aggregation of bacterial cells surrounded
by extracellular polymeric substances (EPS) which are secreted from the cells.
Biofilm has a significant role in chronicity of infection and cells are recalcitrant to
antibacterial agents when biofilm is formed on abiotic or biotic surfaces. Nontypeable
Haemophilus influenzae (NTHi) has been investigated previously in terms of biofilm
production ability. This study examined NTHi biofilm formation using a staining
assay and relationship to pathogenicity, and an assessment of the spatial distribution
of the chemical components by Fourier Transform Infrared (FTIR) spectroscopy.
Firstly, a semi-quantitative microtitre plate (MTP) assay for NTHi biofilm formation
was developed and validated for in vitro formation of biofilm. This assay was used to
evaluate the effect of length of storage time on four fresh NTHi clinical isolates.
Isolates were stored at -80Cº for two, four, eight, 12, 24 and 48 weeks before
measuring biofilm production. Sixty clinical isolates of NTHi from different body
sites were also screened for biofilm formation ability and relationship with the body
site. FTIR microspectroscopy was applied to study NTHi biofilm formation and
pathogenesis by generating FTIR spectra. FTIR spectroscopy coupled with imaging of
the biofilm components were assessed for untreated biofilm, as well as the effect of
antibiotic treatment on new biofilm formation and treatment of mature NTHi biofilm.
The MTP showed consistent differences between the different NTHi isolates tested in
first part of the project with these categorised as high and low biofilm producers. The
MTP assay demonstrated that frozen storage of the isolates was not a determinant of
biofilm formation. No statistically significant differences in the in vitro biofilm
production were found across the clinical NTHi isolates from the nasopharynx
(normal flora), ears (otitis media), lung (community acquired pneumonia and
bronchiectasis in cystic fibrosis) or the isolates of Haemophilus haemolyticus (oropharynx, normal flora). However, the isolates from eyes (conjunctivitis)
demonstrated remarkably consistent low biofilm production compared to the isolates
from other body site (P<0.005). In the second part of this project, FTIR spectroscopy
was used to analyse the chemical constituents of the different biofilms. Unsupervised
multivariate analysis (PCA) of the spectral data showed a chemical distinction
between the two categories of biofilm formation (high and low). Analysis of
microscopic IR hyperspectral data highlighted the spatial distribution of the different
chemical components of the biofilm such as protein and carbohydrate. Analysis of
antibiotic treated biofilm by FTIR showed an increase of protein bands in NTHi
compared to standard Haemophilus influenzae isolate and untreated biofilm.
This project provides detailed information about ability of formation, pathogenesis
and chemical composition of biofilms produced by NTHi isolates. Spectral
information, with the effect of antibiotic on mature and newly formed NTHi biofilms
provides a spatial overview of chemical differences in the bacterial biofilm.

Item Type:	Thesis - PhD
Authors/Creators:	Obaid, NA
Keywords:	NTHi, biofilm formation, FTIR spectroscopy, hyperspectral data, AMX, MIC and sub-MIC
Copyright Information:	Copyright 2015 the Author
Additional Information:	Chapter 3 appears to be the equivalent of a post-print version of an article published as: Obaid, N. A., Jacobson, G. A., Tristram, S., (2015). Relationship between clinical site of isolation and ability to form biofilms in vitro in nontypeable Haemophilus influenzae, Canadian journal of microbiology, 61(3), 243-245. © Copyright 2015 – Canadian Science Publishing
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