High-throughput mutagenesis and screening approach for the identification of drug-resistant mutations in the rifampicin resistance-determining region of mycobacteria.

Rifampicin is the most powerful first-line antibiotic for tuberculosis, which is caused by Mycobacterium tuberculosis. Although accumulating evidence from sequencing data of clinical M. tuberculosis isolates suggested that mutations in the rifampicin-resistance-determining region (RRDR) are strongly associated with rifampicin resistance, the comprehensive characterisation of RRDR polymorphisms that confer this resistance remains challenging. By incorporating I-SceI sites for I-SceI-based integrant removal and utilizing an L5 swap strategy, we efficiently replaced the integrated plasmid with alternative alleles, making mass allelic exchange feasible in mycobacteria. Using this method to establish a fitness-related gain-of function screen, we generated a mutant library that included all single-amino-acid mutations in the RRDR, and identified the important positions corresponding to some well-known rifampicin-resistance mutations (Q513, D516, S522, H525, R529, S531). We also detected a novel two-point mutation located in the RRDR confers a fitness advantage to M. smegmatis in the presence or absence of rifampicin. Our method provides a comprehensive insight into the growth phenotypes of RRDR mutants and should facilitate the development of anti-tuberculosis drugs.

Breadth of knowledge vs. grades: What best predicts achievement in the first year of health sciences programmes?

This study aimed to identify those features within secondary school curricula and assessment, particularly science subjects that best predict academic achievement in the first year of three different three-year undergraduate health professional programmes (nursing, pharmacy, and health sciences) at a large New Zealand university. In particular, this study compared the contribution of breadth of knowledge (number of credits acquired) versus grade level (grade point average) and explored the impact of demographic variables on achievement. The findings indicated that grades are the most important factor predicting student success in the first year of university. Although taking biology and physics at secondary school has some impact on university first year achievement, the effect is relatively minor.