Application of infra-red spectroscopy to the evaluation of biofilm formation and pathogenesis of nontypeable Haemophilus influenzae

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.