The Beginning of therpoBGene 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

Abstract

We aimed to study the distribution and contribution of mutations in the rpoB whole gene in rifampin-resistant/rifabutinresistant (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% ( A ccording to the data reported by the World Health Organization (WHO) in 2010, an estimated 440,000 cases of multidrug-resistant tuberculosis (MDR-TB) (primary and acquired) arose in 2008. Among all incident TB cases globally, 3.6% are estimated to have MDR-TB (19), so a series of new drugs or alternatives for treatment of MDR-TB has been recommended by WHO since 2008 (20). Rifabutin (Rfb) has been one of the most efficacious drugs and the first-line oral agent recommended by WHO for MDR-TB, but Rfb sometimes has cross-resistance to rifampin (RIF) (2, 4, 15), which implies that the characterization of rpoB mutations in Rfb-resistant (Rfb r ) isolates is not completely the same as that of rpoB mutations in RIF-resistant (RIF r ) isolates. There have been few data supporting this conjecture to this day because of a lack of characterization of rpoB mutations in Rfb r clinical Mycobacterium tuberculosis isolates, so it is very hard to identify the Rfb r isolates from RIF r isolates, even though so many molecular assays for prediction of rifampin susceptibility have been established (8,1,13,3,5). Furthermore, phenotypic drug susceptibility testing is too time-consuming, which is a tremendous obstacle for MDR-TB therapy with rifabutin.According to the results of research presented previously, the mechanism of resistance to rifampin and rifabutin is commonly amino acid exchange in the ␤ subunit (rpoB) of the DNA-directed RNA polymerase. So, the rpoB mutations in M. tuberculosis isolates, whose susceptibility to rifampin, rifabutin, and isoniazid had been previously determined, were characterized after their rpoB whole genes were sequenced.

Role of pncA and rpsA Gene Sequencing in Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from Southern China

“…This was similar to other reports (23-25) and similar to the "all pncA mutations" collected and analyzed in the TB Drug Resistance Database (http://www .tbdreamdb.com/PZA_Rv2043c_AllMutations.html), in which mutations were randomly distributed from nt 1 to nt 554. Only 13 of 44 (29.5%) mutants in this study located in the clustering of mutations of G132-T142 and P69-L85 found by previous investigators (25), which showed that the location of mutations in the pncA gene was more variable than expected before (25) and that the mutations were not clustered as in other genes associated with drug resistance, such as the rpoB gene (12). None of the isolates with identical pncA mutations were epidemiologically linked, so the PZA-resistant strains might not have been obtained from transmission.…”

Next-Generation Ion Torrent Sequencing of Drug Resistance Mutations in Mycobacterium tuberculosis Strains

“…Using LPA, these 6 strains were determined to be resistant, since both mutant and wild-type bands were absent ( Table 2). The D516G mutation occurs less frequently than D516V, is not clustered or epidemiologically significant, and has been reported to occur in isolates characterized from China, Africa, and North America (7,12,23). Similarly, an uncommon amino acid substitution was identified at position 526 in the rpoB gene product.…”

Rapid, High-Throughput Detection of Rifampin Resistance and Heteroresistance in Mycobacterium tuberculosis by Use of Sloppy Molecular Beacon Melting Temperature Coding

“…The three-point T m code produced by our assay either specifically identified RRDR mutations or grouped them into easily identifiable mutation clusters. This ability to subtype different RRDR mutations may prove useful in epidemiological investigations or where different rifamycins confer different levels of resistance (20,28,30,35). The assay did not depend on high-resolution T m capabilities.…”