Abstract

This project aimed to investigate the application of Matrix-Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS) for the identification and phenotypic characterisation of difficult to culture microbes. The main bulk of the work focussed on the rapid identification of Acanthamoeba, a parasite which can cause a severe eye infection (keratitis) that can lead to blindness if not quickly diagnosed. Through this project, a method for generating useful MALDI spectra for Acanthamoeba has been developed, with the identification of six peaks that are common to all 15 isolates used. These peaks do not co-identify with any other species in the SARAMIS (Spectral ARchive And Microbial Identification System) database. Currently there are no clinically useful MALDI spectra for the identification of Acanthamoeba. The sensitivity of generating useful MALDI spectra for Acanthamoeba have been determined to be 4x105 cells/ml; which is greater than the sensitivity required for bacterial identification via MALDI (1x104). Also, data generated during this project indicates that MALDI may differentiate to genotype level. This is important as the nomenclature and speciation of Acanthamoeba has moved away from species names to genotypes.
In addition, the potential of MALDI for determining bacterial isolate antibiotic sensitivity was investigated. This was focussed on the expression of β–lactamase enzymes, and for this a number of plasmid systems were utilised. Although, this part of the research work couldn’t achieve its initial aims of detecting the enzymes, however, the impact of plasmid acquisition on the MALDI spectrum of isolates was observed. Spectra changed with the level of plasmid induced expression in isolates and this is likely due to alteration in the profile of proteins expressed; this is the first observation of this effect. It is known that large scale industrial fermentation systems often show decreased product expression under stress and in some cases undergo plasmid ‘curing’. The cost of these events is so significant thus a rapid method for monitoring expression within cells would be a step forward in continuous process monitoring; MALDI may provide such a method.