| The high epidemicity and infectivity of tuberculosis (TB) is a great challenge to public health. TB is classified by WHO as one of the infection diseases which need to control carefully. It is said that one-third of the world's population is currently infected with the TB bacillus and someone in the world is newly infected with TB bacilli every second (WHO, 2006). Mycobacterium is belonging to the genus Mycobacterium, mycobacteriaceae, actinomycetales. Mycobacterium tuberculosis (M. tuberculosis) is pathogenic in clinic, and some non-tuberculous mycobacteria (NTM) also are pathogenic. It is difficult to identify the patients with clinical lung disease caused by NTM or TB only from the pathological changes. But different species display different antibiotic resistances and treatment prescriptions. It is important to identify Mycobacteria at the species level promptly and accurately for control the epidemic situation.Mycolic acids (MAs) are unusually high lipid content in cell wall of mycobacteria. They are stable high-molecular-weightα-branched,β-hydroxy fatty acids with fingerprinted feature. These fatty acids are differed in their chromatographic behaviors from the chain lengths, the degree of unsaturations and other functional groups. Because of the relationship between composition of MAs and bacteria species, different species of mycobacterium can provide different fingerprinted feature.A reversed phase high-performance liquid chromatography (RP-HPLC) method was developed to identify Mycobacterium species and construct a fingerprints library in this paper. This approach provides an alternative method with rapidity, highly reliability for the identification of Mycobacterium by analysis the differences of mycolic acids. This work is also important to provide a technology and an additional library.This dissertation consists of four chapters:Chapters one was the preface of the dissertation. The recent developments of identification methods and typing techniques, the problems of these methods were briefly described. The application of high performance liquid chromatography (HPLC) as a modern identification technique was emphasized. The research proposals and contents to be carried out were brought forward based on the present difficulties.In chapter two, a curvilinear gradient RP-HPLC method using methanol and methylene chloride as mobile phase was developed. MAs from each culture of Mycobacterium species were extracted, saponified, acided, derivatized and analyzed by the RP-HPLC method. This approach provides an alternative method with rapidity, highly reliability for the identification of Mycobacterium.In chapter three, based on the method which set up by chapter two, a cultured mycobacteria mycolic acids fingerprints library of HPLC patterns was constructed, including 49 species (come from ATCC) which recorded in'Bergey's manual of systematic bacteriology'. Based on the distribute feature of the peaks, the initial step for identifying a species is determining the number of peak clusters. These patterns can be classified to three types, such as single-cluster (14), double-clusters (23), and triple- and multi-clusters (12). Then, the identification of Mycobacterium species was deduced from the HPLC patterns of relative retention time and the relative peak height. 41 species have been successfully identified according to this method (the other 8 species were difficult to identify accurately by this method). A supplementary GC method was established to try to identify some difficult identified mycobacteria.In chapter four, based on the above work, we analyszed a great many mycobactia which come form DSMZ (including the strains which not recorded in'Bergey's manual of systematic bacteriology') mycolic acids by HPLC. These fingerprints are important to provide a technology and an additional library for typing the mycobacteria.
|
PDF Full Text Request