Studies of Polymorphic DNA Fingerprinting and Lipid Pattern of Mycobacterium tuberculosis Patient Isolates in Japan

Abstract

Strain differentiation by DNA restriction fragment length polymorphism (RFLP) has been used mainly for the epidemiological purpose of Mycobacterium tuberculosis infection. In this study, we tried to connect the molecular and phenotypic characteristics of M. tuberculosis patient isolates by comparing the DNA fingerprints obtained by RFLP using IS6110 and lipid patterns using two‐dimensional thin‐layer chromatography (2‐D TLC) with silica gel, since M. tuberculosis has a lipid‐rich cell envelope which contributes to the virulence and immunomodulatory properties. We found that 66 isolates of M. tuberculosis from tuberculosis patients showed that the occurrence of IS6110 varied from 1 to 24 copies. The IS6110 patterns were highly variable among isolates. Fifty different RFLP patterns were observed, and 12 RFLP patterns were shared by two or more strains. By computerized analysis of the RFLP patterns of M. tuberculosis patient isolates, we found that 95% of the isolates fell into seven clusters, from A to G, with at least two isolates in each (> 30% similarity). Among the cellular lipids, the phospholipid composition did not differ by strain, whereas the glycolipid pattern differed markedly. Especially, the relative concentration of cord factor and sulfolipid, both of which were known as virulent factors, varied by strain. The fingerprints of some strains showed an association between the DNA and glycolipid patterns, even though some of the same DNA fingerprint strains showed differences in lipid patterns. Among the patient isolates, M. tuberculosis strain 249 possessed a specific glycolipid with 2‐O‐methyl‐L‐rhamnose and L‐rhamnose, which is rarely found in other strains. This glycolipid showed serological activity against the sera of tuberculosis patients, even if the reactivity was not as strong as trehalose dimycolate. It also showed the inhibition of phagosome‐lysosome fusion in macrophages, suggesting involvement with virulence. These results suggest that RFLP analysis using IS6110 is useful for clustering the human isolates of M. tuberculosis, however, for further strain differentiation on virulence, a lipid analysis provides more information.

Rapid Serodiagnosis of Active Pulmonary Mycobacterium tuberculosis by Analysis of Results from Multiple Antigen-Specific Tests

“…In our study, the percentages of patients who were positive in all three tests were not high, even in patients with smear-positive and chronic active pulmonary TB. The antibody response in these patients, who had experienced long-term and heavy exposure to bacilli, was more diverse than we had expected based on previous reports (4,20). Furthermore, when the results of a single test were supplemented with results from other tests, the detection rate increased to 91.5% in the active pulmonary TB group.…”

“…The cell wall antigen compositions of the tuberculous bacilli differ among isolates (4,9). The TB patient does not produce antibodies against all antigenic substances in the cell walls of the tuberculous bacilli, and the specificities of the antibodies differ among patients.…”

“…The combined use of trehalose-dimycolate and minor glycolipids in the TBGL assay, rather than purified trehalose-dimycolate alone, results in increased diagnostic sensitivity for TB (13). The cell wall antigen composition of each patient isolate of tuberculous bacilli differs, resulting in antibodies with different specificities among patients (4,9). We hypothesized that the TB patient does not produce antibodies against all antigenic substances in the cell wall of the tuberculous bacilli and that the specificities of the antibodies differ among patients.…”

Clinical Evaluation of Anti-Tuberculous Glycolipid Immunoglobulin G Antibody Assay for Rapid Serodiagnosis of Pulmonary Tuberculosis

“…(ii) Sensitivity differs depending upon the differences in composition of the mycolic acid subclasses with regard to the antigenicity of TDM (17). (iii) The glycolipid composition of the tuberculous bacillus isolated from patients was reported to differ in each patient (7). In terms of the diagnosis of tuberculosis, however, patients who were identified as tuberculosis positive by smear test or by the nucleic acid amplification test did not need to undergo serodiagnosis.…”