Identifying Genetic Factors Promoting Fitness and Rifampicin Tolerance in Mycobacterium Tuberculosis Biofilms

OBJECTIVE: In 2011, the World Health Organization reported 8.7 million new cases of tuberculosis and 1.4 million tuberculosis-related deaths worldwide. The causative agent of this disease Mycobacterium tuberculosis is a notoriously persistent pathogen whose treatment requires a 6-9 month course of multiple antibiotics to clear the infection. Here, we used next generation DNA sequencing to identify several genetic factors involved in the tolerance of M. tuberculosis to the antibiotic rifampicin in the context of the biofilm, a common bacterial stress survival strategy.\ud \ud METHODS: A transposon insertion mutant library was constructed in the Erdman strain of M. tuberculosis. The library was then grown planktonically or in a biofilm and exposed to 5μg/ml of rifampicin. After exposure samples were processed and plated for colony formation. DNA was extracted from the colonies and prepared for sequencing by PCR amplification of transposon junction sites. DNA of these sites was sequenced using Illumina Hi-Seq technology and analyzed to find differential representation of transposon mutants between the unexposed and exposed library samples.\ud \ud RESULTS: Biofilms of M. tuberculosis are more tolerant than planktonic samples in vitro to rifampicin treatment. Seven genes at eight genomic positions were found to contain insertion sites that were ubiquitous in the samples unexposed to rifampicin, but found to be greatly decreased in the exposed biofilm samples: Rv0385, Rv1508c (at two positions), Rv1819c, Rv2779c, Rv3164c, Rv3796 and Rv3868.\ud \ud DISCUSSION: Further analysis of these mutants by using knockouts and studying the effects in vitro or in a mouse model will be necessary to confirm their role in rifampicin tolerance in an actual infection and determine any possible clinical benefits in exploiting these genetic factors. As latent infection remains a significant problem in global public health, exploitation of targets that contribute the persistence of infection could be a valuable tool in clearing infections