Khatri, Bhagwati (2010) Metabolic phenotype of bovine tuberculosis field strains. (MSc(R) thesis), Kingston University, .

Abstract

Whilst the whole genome sequences of M tuberculosis H37 Rv, M bovis AF6112122/97 and BCG have been published and detailed genetic analysis of differences between Mycobacterium tuberculosis complex (MTBC) strains have been explored, little of this knowledge has been related to phenotypes. The current study employs the high throughput metabolic assay Biolog Phenotype MicroArray (PM) technology has been used for the first time to examine phenotypic differences in slow growing mycobacteria. Two established methods MTT (Tetrazolium dye) and Oxygen Electrode (Measuring oxygen consumption) were used to validate PM technology and previous studies on metabolism of mycobacteria using these existing methods were reviewed to put the current work into context. For PM technology to be useful background reading had to be taken into account. Systematic investigations of dye reduction and starvation revealed that a 24 hour starvation step succeeded in diminishing background dye reduction. Utilisation of carbon substrates by M tuberculosis H37 Rv was significantly different to M bovis type 9 strains. M tuberculosis H37 Rv used 7 substrates not used by M bovis AF6112122/97 while M bovis AF6112122/97 used 4 substrates not used by M tuberculosis H37 Rv. Interestingly, a few differences were also observed within type 9 strain panel which showed the utilisation of Tween 80, Monomethyl succinate, Glycerol and D-malate to be significant different between the strains in the panel. Although metabolism of known nitrogen sources such as L-alanine and D-serine were confirmed by MTBC strains, utilisation of many amino acids for which auxotrophs have been obtained for M tuberculosis H37 Rv was not shown in this work. These data suggest a requirement for, and indeed may assist in the optimisation of the PM technology for MTBC. This work represents the first step in using PM technology to provide applied, practical information that can be implemented in the diagnostic arena, principally in facilitating the improvement of current media to ensure good growth of all strains.

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