Disparities in Capreomycin Resistance Levels Associated with the rrs A1401G Mutation in Clinical Isolates of Mycobacterium tuberculosis

Abstract

bAs the prevalence of multidrug-resistant and extensively drug-resistant tuberculosis strains continues to rise, so does the need to develop accurate and rapid molecular tests to complement time-consuming growth-based drug susceptibility testing. Performance of molecular methods relies on the association of specific mutations with phenotypic drug resistance and while considerable progress has been made for resistance detection of first-line antituberculosis drugs, rapid detection of resistance for secondline drugs lags behind. The rrs A1401G allele is considered a strong predictor of cross-resistance between the three second-line injectable drugs, capreomycin (CAP), kanamycin, and amikacin. However, discordance is often observed between the rrs A1401G mutation and CAP resistance, with up to 40% of rrs A1401G mutants being classified as CAP susceptible. We measured the MICs to CAP in 53 clinical isolates harboring the rrs A1401G mutation and found that the CAP MICs ranged from 8 g/ml to 40 g/ml. These results were drastically different from engineered A1401G mutants generated in isogenic Mycobacterium tuberculosis, which exclusively exhibited high-level CAP MICs of 40 g/ml. These data support the results of prior studies, which suggest that the critical concentration of CAP (10 g/ml) used to determine resistance by indirect agar proportion may be too high to detect all CAP-resistant strains and suggest that a larger percentage of resistant isolates could be identified by lowering the critical concentration. These data also suggest that differences in resistance levels among clinical isolates are possibly due to second site or compensatory mutations located elsewhere in the genome. Global tuberculosis (TB) control efforts are severely complicated by the emergence and spread of multidrug-resistant (MDR) TB, as evidenced by the half million new MDR TB cases reported by the World Health Organization (WHO) last year (1). MDR TB is defined as Mycobacterium tuberculosis resistant to two of the most potent first-line antituberculosis agents, rifampin (RIF) and isoniazid (INH) (1). Treatment requires the administration of second-line antibiotics that are more expensive, can lead to more adverse side effects, and increase treatment duration time relative to first-line drugs (1, 2). Unfortunately, inadequate treatment of MDR TB and the misuse of second-line antibiotics have contributed to the emergence of extensively drug-resistant (XDR) TB strains, defined by WHO as multidrug resistant with additional resistance to any fluoroquinolone and at least one of the injectable aminoglycoside agents kanamycin (KAN), amikacin (AMK), or capreomycin (CAP) (1).Rapid and reliable diagnosis of antibiotic resistance is critical in controlling the spread of drug-resistant TB. Laboratories are increasingly adopting rapid molecular methods to identify mutations associated with resistance as a complement to the more time-consuming growth-based methods (3, 4). Though molecular methods can increase specificity and drastically reduce delay in th...

Molecular Investigation of Resistance to Second-Line Injectable Drugs in Multidrug-Resistant Clinical Isolates of Mycobacterium tuberculosi s in France

“…Thus, AMK resistance in these 2 isolates can be regarded as borderline, and one cannot exclude that the 2 strains would have been ranked as S-AMK if a higher AMK concentration had been used on primary DST (30 mg/liter according to the WHO 2014 guidelines) (33). One should note that the WHO provides no evidence based on which the recommended critical concentrations have been set, a point that has to be considered when explaining the discrepancies between genotype and phenotype, as previously suggested (9,34).…”

“…The DST was repeated, and MICs were determined on Middlebrook 7H10 plates (33) containing KAN at 0.625, 1.25, 2.5, 5, 10, 20, and 40 mg/liter or AMK and CAP at 0.5, 1, 2, 4, 8, 16, and 32 mg/liter for all the strains with unexpected combinations of resistances and mutations (and not explained by a low percentage of resistant mutants that were not detected by sequencing of drug resistance-associated genes on the primary culture but were detected by sequencing from tubes with antibiotics). The MIC was defined as the lowest concentration of drug resulting in growth of Յ1% of the initial inoculum after 4 weeks of incubation at 37°C (9). The reference strain M. tuberculosis H37Rv (ATCC 27294), which is sensitive to all the drugs tested in our experiment, was included as a control strain.…”

“…Cross-resistance to second-line injectable drugs (AMK, KAN, and CAP) is known to be caused by mutations at positions 1401, 1402, and 1484 in the rrs gene encoding 16S rRNA with the following expression patterns: rrs substitution A1401G displays CAP resistance (R-CAP), with disparities in resistance levels, and high-level resistance to AMK and KAN; the rrs C1402T substitution displays low-level resistance to KAN and high-level resistance to CAP and retains susceptibility to AMK (S-AMK); and the rrs G1484T substitution displays high-level resistance to all 3 drugs (8,9). However, these mechanisms in rrs have never been formally demonstrated by allelic exchange data.…”

Validation of novelMycobacterium tuberculosisisoniazid resistance mutations not detectable by common molecular tests

“…pJV128 (33) was provided by Graham F. Hatfull (University of Pittsburgh). All mycobacterial broth cultures were grown as described previously (34). This work was approved by the CDC Institutional Review Board (protocol 5411).…”

“…H37Rv carrying the pJV128 recombineering plasmid was generated previously (34) and was used as a recipient strain in recombineering experiments as described therein, with some modifications. 7H9-succinate broth was prepared as described in (38) but cyclohexamide and carbenicillin were omitted.…”