The beginning of the rpoB gene in addition to the rifampin resistance determination region might be needed for identifying rifampin/rifabutin cross-resistance in multidrug-resistant Mycobacterium tuberculosis isolates from Southern China.

We aimed to study the distribution and contribution of mutations in the rpoB whole gene in rifampin-resistant/rifabutin-resistant (RIF(r)/Rfb(r)) (or RIF/Rfb cross-resistant) clinical Mycobacterium tuberculosis isolates. One standard M. tuberculosis strain (H37Rv) and 392 other clinical M. tuberculosis isolates mainly from Guangdong Province of China whose susceptibilities to rifampin (RIF), rifabutin (Rfb), streptomycin (SM), ethambutol (EMB), and isoniazid (INH) were previously determined were subjected to DNA sequencing of their rpoB whole genes. H37Rv and the 30 drug-susceptible clinical isolates had no mutations in rpoB whole genes. In 43 rifampin-resistant/rifabutin-susceptible (RIF(r)/Rfb(s)) isolates, the most frequent mutation codons were 516 (62.80%), 526 (14.0%), and 533 (6.98%), but codon 531 had no mutation. Twenty-one of the 43 isolates (48.84%) had single mutations of H526L, H526S, D516V, D516Y, and D516F. In 319 RIF(r)/Rfb(r) isolates, the most frequent mutation codons were 531 (73.7%) and 526 (18.8%); the mutation frequency for codon 516 was 2.5%, and that for codon 533 was only 0.31%. A total of 82.8% (264/319) of them had single mutations of S531L, S531W, H526D, H526Y, H526R, Q513K, Q513P, Q510H, V176F, P206(T)R, Y314(T)C, and H323(T)Y (the superscript T indicates M. tuberculosis numbering; the remaining codons use the E. coli numbering), among which V176F, P206(T)R, Y314(T)C, and H323(T)Y were located in the beginning of rpoB, and all of them were present in 1.9% (6/319) of RIF(r)/Rfb(r) isolates. The multiple mutations in RIF(r)/Rfb(r) isolates and in RIF(r)/Rfb(s) isolates were also different from each other either in mutation positions or in types of mutation combinations. In conclusion, the mutations of rpoB in RIF-R/Rfb(s) and in RIF-R/Rfb-R isolates differ significantly from each other not only in the most frequent mutation codons (516, 531, and 533) but also in the most frequent single mutations (S531L, H526L, D516V, D516Y, and D516F), and the beginning of rpoB may confer a RIF/Rfb cross-resistance phenotype in M. tuberculosis. Molecular assays for identifying RIF/Rfb cross-resistance in M. tuberculosis might be improved in terms of accuracy by including this region, in addition to the rifampin resistance determination region.