Genetic Determinants Involved in p -Aminosalicylic Acid Resistance in Clinical Isolates from Tuberculosis Patients in Northern China from 2006 to 2012

Abstract

b p-Aminosalicylic acid (PAS) is an important compound for treating multidrug-resistant tuberculosis (TB). Previous studies showed that thyA mutations are often related to PAS resistance in clinical isolates. We performed a systematic analysis of isolate genotypes and detected mutations in three folate pathway genes (folC, thyA, and ribD) in 61.1% (127/208) of PAS-resistant isolates, including 11 double mutants. This result expands our knowledge about the distribution and frequency of mutations related to PAS resistance in mycobacterial clinical isolates.T uberculosis (TB) is a major global health problem that affected 8.6 million new patients and caused 1.3 million deaths worldwide in 2012 (1). The prevalence of multidrug-resistant TB resistant to isoniazid and rifampin poses a great challenge to TB control and necessitates more complicated and longer treatment regimens. The compound p-aminosalicylic acid (PAS), one of the first effective antibiotics for treating TB, remains a primary component of many multidrug-resistant TB therapies (2, 3).PAS is a prodrug that targets the folate pathway of Mycobacterium tuberculosis (4). Mutations in the dihydrofolate synthase FolC, which is required for the bioactivation of PAS, confer PAS resistance (5). In addition, mutations causing an overexpression of RibD, which can act as an alternative dihydrofolate reductase (DHFR/DfrA), confer resistance (5, 6). Finally, loss-of-function mutations in thymidylate synthase ThyA, which is a major consumer of tetrahydrofolate, result in PAS resistance (7,8).Other studies reported that one-third of PAS-resistant isolates had thyA mutations; however, little is known about the antibiotic resistance mechanism(s) in the remaining samples (9, 10). Here, we first randomly selected PAS-resistant isolates to determine their genotypes for folC, thyA, and ribD. Two hundred eight isolates were selected from the Beijing Bio-Bank of Clinical Resources on Tuberculosis, which included isolates from approximately 6,400 patients from 2006 to 2012. These patients received routing clinical care and were from provinces and regions in northern China, most of whom were undergoing retreatment or had relapsed. Drug susceptibility testing (DST) for 10 anti-TB drugs was performed with these isolates according to World Health Organization guidelines (11). This study was approved by the ethics review committee of the Institute of Pathogen Biology, Chinese Academy of Medical Sciences, and Peking Union Medical College.Resistance to PAS was determined by cultivating bacteria in the presence of the critical concentration of 1.0 mg/liter PAS on Lowenstein-Jensen medium (12). All isolates were putatively identified as M. tuberculosis by 16S rRNA gene sequencing (13). Molecular characterization of the TB strains was performed by the spoligotyping method based on M. tuberculosis-specific direct repeat (DR) region (14).Sequencing analysis of the PAS resistance-related genes from the isolates was performed as previously described (10). Briefly, the coding and flanking regi...

Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis

“…However, our analysis highlighted that Rv1482c-fabG1 and inhA mutations, in the absence of katG S315T, can emerge prior to MDR-TB, as previously shown in two phylogenetically-independent clades in Lisbon 16,17 The gene-based GWAS of XDR-TB versus MDR-TB identified mutations in gyrA (FLQ), rrs (aminoglycosides), the embC-embA intergenic region and ubiA (EMB). The PhyC test additionally revealed eis-Rv2417c (KAN), gyrB (FLQ), rrs (aminoglycosides), folC (PAS), alr (CYS), gid (STM) SNPs, and a novel mutation in the thyX-hsdS.1 intergenic region (A-9T, PAS) 18,19 . In addition to loci identified above, the gene-based GWAS comparing XDR-TB to susceptible groups identified rpoC (a compensatory mechanism for RIF resistance), ethA (ETH), eis-Rv2417c (KAN) and PPE52-nuoA (novel intergenic region, G-314T).…”

Targeting intracellular p-aminobenzoic acid production potentiates the anti-tubercular action of antifolates

“…Mutations in folC , encoding dihydrofolate synthase, confer PAS resistance in both laboratory and clinical isolates182122. We deleted pabB in a previously described PAS resistant folC mutant containing a glutamate to alanine substitution at position 153 ( folC E153A )22.…”

Antibiotic resistance mechanisms in M. tuberculosis: an update

“…Most acquired PAS resistance mutations in M. tuberculosis have thus far been mapped to thyA , folC , dfrA and ribD (Table 2) (Mathys et al 2009; Zhang et al 2015; Zhao et al 2014; Zheng et al 2013). …”