Chemical Genetic Interaction Profiling Reveals Determinants of Intrinsic Antibiotic Resistance in Mycobacterium tuberculosis.

Chemotherapy for tuberculosis (TB) is lengthy and could benefit from synergistic adjuvant therapeutics that enhance current and novel drug regimens. To identify genetic determinants of intrinsic antibiotic susceptibility in Mycobacterium tuberculosis, we applied a chemical genetic interaction (CGI) profiling approach. We screened a saturated transposon mutant library and identified mutants that exhibit altered fitness in the presence of partially inhibitory concentrations of rifampin, ethambutol, isoniazid, vancomycin, and meropenem, antibiotics with diverse mechanisms of action. This screen identified the M. tuberculosis cell envelope to be a major determinant of antibiotic susceptibility but did not yield mutants whose increase in susceptibility was due to transposon insertions in genes encoding efflux pumps. Intrinsic antibiotic resistance determinants affecting resistance to multiple antibiotics included the peptidoglycan-arabinogalactan ligase Lcp1, the mycolic acid synthase MmaA4, the protein translocase SecA2, the mannosyltransferase PimE, the cell envelope-associated protease CaeA/Hip1, and FecB, a putative iron dicitrate-binding protein. Characterization of a deletion mutant confirmed FecB to be involved in the intrinsic resistance to every antibiotic analyzed. In contrast to its predicted function, FecB was dispensable for growth in low-iron medium and instead functioned as a critical mediator of envelope integrity.

Acetate Dissimilation and Assimilation in Mycobacterium tuberculosis Depend on Carbon Availability.

UNLABELLED: Mycobacterium tuberculosis persists inside granulomas in the human lung. Analysis of the metabolic composition of granulomas from guinea pigs revealed that one of the organic acids accumulating in the course of infection is acetate (B. S. Somashekar, A. G. Amin, C. D. Rithner, J. Troudt, R. Basaraba, A. Izzo, D. C. Crick, and D. Chatterjee, J Proteome Res 10:4186-4195, 2011, doi:http://dx.doi.org/10.1021/pr2003352), which might result either from metabolism of the pathogen or might be provided by the host itself. Our studies characterize a metabolic pathway by which M. tuberculosis generates acetate in the cause of fatty acid catabolism. The acetate formation depends on the enzymatic activities of Pta and AckA. Using actyl coenzyme A (acetyl-CoA) as a substrate, acetyl-phosphate is generated and finally dephosphorylated to acetate, which is secreted into the medium. Knockout mutants lacking either the pta or ackA gene showed significantly reduced acetate production when grown on fatty acids. This effect is even more pronounced when the glyoxylate shunt is blocked, resulting in higher acetate levels released to the medium. The secretion of acetate was followed by an assimilation of the metabolite when other carbon substrates became limiting. Our data indicate that during acetate assimilation, the Pta-AckA pathway acts in concert with another enzymatic reaction, namely, the acetyl-CoA synthetase (Acs) reaction. Thus, acetate metabolism might possess a dual function, mediating an overflow reaction to release excess carbon units and resumption of acetate as a carbon substrate. IMPORTANCE: During infection, host-derived lipid components present the major carbon source at the infection site. ß-Oxidation of fatty acids results in the formation of acetyl-CoA. In this study, we demonstrate that consumption of fatty acids by Mycobacterium tuberculosis activates an overflow mechanism, causing the pathogen to release excess carbon intermediates as acetate. The Pta-AckA pathway mediating acetate formation proved to be reversible, enabling M. tuberculosis to reutilize the previously secreted acetate as a carbon substrate for metabolism.

Characterization of rough and smooth morphotypes of Mycobacterium abscessus isolates from clinical specimens.

Mycobacterium abscessus, which consists of the two subspecies M. abscessus subspecies abscessus and M. abscessus subspecies bolletii, can produce rough or smooth colony morphologies. Here we analyzed 50 M. abscessus isolates cultured from the respiratory specimens of 34 patients, 28 (82%) of whom had cystic fibrosis (CF), with respect to their colony morphologies and antibiotic susceptibilities. The overall proportions of occurrences of the two morphotypes were similar, with specimens from 50% of the patients showing a rough and 38% showing a smooth morphotype. A total of 12% of the specimens from the patients showed both morphotypes simultaneously. At the subspecies level, the proportions of rough and smooth morphotypes differed substantially; 88% of rough morphotypes belonged to M. abscessus subspecies abscessus, and 85% of smooth morphotypes belonged M. abscessus subspecies bolletii. Inducible clarithromycin resistance due to the Erm(41) methylase, as well as high-level resistance to clarithromycin due to mutations within the rrl gene, occurred independently of the morphotype. The MIC50s of amikacin and cefoxitin were identical for the two morphotypes, whereas the MIC50s of tigecycline were 0.25 µg/ml for the rough morphotype and 2.0 µg/ml for the smooth morphotype. Our results show that the smooth morphotype was more dominant in respiratory specimens from CF patients than previously thought. With respect to resistance, colony morphology did not affect the susceptibility of Mycobacterium abscessus to the first-line antibiotics clarithromycin, amikacin, and cefoxitin.