Patterns of rpoB, rpoC, and pncA mutations in drug-resistant Mycobacterium tuberculosis isolated from patients in South Korea

Abstract

Background Rifampin (RIF) is one of the primary first-line combination antibiotics indicated for Mycobacterium tuberculosis, which greatly reduces the length of chemotherapy. Pyrazinamide (PZA) is also an antimicrobial agent, especially effective against multi-drug-resistant (MDR) tuberculosis (TB), resistant to isoniazid (INH) and RIF. M. tuberculosis acquires resistance to RIF through mutations in the rpoB gene, while compensatory mutations in the rpoC gene restore the fitness of RIF-resistant M. tuberculosis. M. tuberculosis acquires its resistance to PZA by having mutations in the pncA gene. A total of 93 M. tuberculosis isolates attained from patients were analysed to examine the mutation patterns of rpoB, rpoC, and pncA in South Korea. Methods Antibiotic susceptibility was determined by carrying out bacterial cultures of drug-resistant mycobacterial isolates. Mutations in the rpoB, rpoC and pncA genes were identified by sequencing analysis, while the attributes of mutations were determined by comparing a relevant wild-type DNA sequence with that of a mutant allele. (H37Rv, American Type Culture Collection 25618). Results A drug susceptibility test was performed for the total of 93 M. tuberculosis isolates that had been successfully cultured. Of these 93 isolates that were subjected to drug susceptibility testing (DST), 75 were found to be resistant to multiple drugs. Of these 75 isolates, 20 were MDR-TB; 7 were MDR-Plus; 36 were extensively drugresistant XDR-TB; and 12 were drug-resistant (DR)-TB. A total of 66 cultured M. tuberculosis isolates were found to be RIF-resistant; 40 cultured isolates were found to be PZA-resistant; 39 cultured isolates were found to be both RIF- and PZA-resistant; and 18 were identified as being pan-susceptible (pan-S). Substitutions or multiple-site mutations in the rpoB region were identified in 56 isolates (56/80, 70.0%), of which 91.1% (51/56) were resistant to RIF and 9 distinctive-site mutations were identified. Fifteen (15) different types of rpoC mutations were identified in 24 isolates (24/93, 25.8%), all of which were resistant to both INH and RIF. The mutation rates in MDRand XDR-TB were 37.0% (10/27) and 38.9% (14/36), respectively. Substitutions of a single nucleotide (22/24, 91.7%) or substitutions of multiple-site mutations (2/24, 8.3%) in the rpoC region were identified, and neither deletion nor insertion mutation was detected in any of the isolates. No mutations were identified in the rpoC region of any drug-susceptible strains. Various mutations were identified in the pncA gene in 46 isolates: Nucleotide substitutions, deletions, insertion, multiple-site mutations and 25 different mutation sites were found. Of these various mutations detected in 46 isolates, substitution of a single nucleotide was most common (27/46, 58.7%), followed by multiple-site mutations (4/46, 8.7%) and insertion (4/46, 8.7%). Frameshifts caused by an insertion or a deletion of a single or multiple nucleotides at various sites accounted for 15.2% (7/46) of all mutations. Conclusion Mutations of the rpoB, rpoC and pncA genes are the essential mechanism of RIF and PZA resistance in drug-resistant M. tuberculosis isolates. Detection of rpoB, rpoC and pncA gene mutations can complement in vitro DST and DNA-based diagnosis of RIF and PZA resistance, and is a promising method for the rapid detection of drug resistance.