Genomic Sequence Analysis of Mycobacterium tuberculosis H37Rv and M. tuberculosis DR-689 Subjected to Continuous Subculturing
Creators
- 1. Universidad Autonoma de Nuevo Leon
- 2. Servicio de Dermatologia Universidad Autonoma de Nuevo Leon Monterrey Mexico
Description
Background: Tuberculosis is caused by the bacterium Mycobacterium tuberculosis, considered as a re-emergent infectious disease and a major public health problem affecting one third of earth’s population. The only vaccine available is BCG, an attenuated M. bovis isolate obtained by continous subculture. Results after using this vaccine on human populations are quiet variable. One of the explanations is that it is not an M. tuberculosis sensu stricto. In order to explore the biological and genetic changes produced in M. bovis that ended in BCG, we have subjected several M. tuberculosis isolates to continuous growth for several years. Here we present, the results of cytotoxicity assay on THP-1 macrophage monolayers as well as the genomic sequence analysis data after 200 subcultures of two of the, M. tuberculosis H37Rv and M. tuberculosis DR-689, a Beijing strain.
Methods: Strains were subcultured in M7H9 broth with 0.5% ox bile in 50 ml Erlenmeyer flasks and incubated at 37?C /150 rpm until observation of visible growth. To measure virulence, the ability of isolates to destroy a THP-1 macrohage monolayer was tested. The WGS was obtained by using an Illumina genome analyzer IIx. The reads were aligned to the reference genome M. tuberculosis H37Rv and a list of polymorphisms -SNPs (single nucleotide polymorphisms) and INDELs (insertions and deletions) - was obtained.
Results: Ability of subcultured isolates to produce cittotoxicity to the THP-1 macrophages was decreased almost at the level of the M. bovis BCG. As controls we used the non-subcultured isolate and M. bovis BCG. More than 100 SNP`s (that is the amount enough to consider individual isolates) between the parental and subcultured isolates were observed in each case; genetic changes affecting putative virulence genes were observed.
Conclusions: As in M. bovis BCG, we observed that continuous subculturing of M. tuberculosis induces important biological changes. Data obtained from the re-sequencing analysis support our previous results concerning the lipid patterns and cytotoxicity.
Supported by CONACYT 155892.
Files
Files
(8.0 MB)
Name | Size | Download all |
---|---|---|
md5:2b17990eeaf45d54bd357547fc274939
|
8.0 MB | Download |