Association between 16S rRNA gene mutations and susceptibility to amikacin in Mycobacterium avium Complex and Mycobacterium abscessus clinical isolates

Abstract

We evaluated the association between 16S rRNA gene (rrs) mutations and susceptibility in clinical isolates of amikacin-resistant nontuberculous mycobacteria (NTM) in NTM-pulmonary disease (PD) patients. Susceptibility was retested for 134 amikacin-resistant isolates (minimum inhibitory concentration [MIC] ≥ 64 µg/ml) from 86 patients. Amikacin resistance was reconfirmed in 102 NTM isolates from 62 patients with either Mycobacterium avium complex-PD (MAC-PD) (n = 54) or M. abscessus-PD (n = 8). MICs and rrs mutations were evaluated for 318 single colonies from these isolates. For the 54 MAC-PD patients, rrs mutations were present in 34 isolates (63%), comprising all 31 isolates with amikacin MICs ≥ 128 µg/ml, but only three of 23 isolates with an MIC = 64 µg/ml. For the eight M. abscessus-PD patients, all amikacin-resistant (MIC ≥ 64 µg/ml) isolates had rrs mutations. In amikacin-resistant isolates, the A1408G mutation (n = 29) was most common. Two novel mutations, C1496T and T1498A, were also identified. The culture conversion rate did not differ by amikacin MIC. Overall, all high-level and 13% (3/23) of low-level amikacin-resistant MAC isolates had rrs mutations whereas mutations were present in all amikacin-resistant M. abscessus isolates. These findings are valuable for managing MAC- and M. abscessus-PD and suggest the importance of phenotypic and genotypic susceptibility testing.

Treatment outcomes of Mycobacterium avium complex pulmonary disease according to disease severity

“…NTM species were identified using polymerase chain reaction-restriction fragment length polymorphism analysis or reverse-blot hybridization of the rpoB gene. Beginning in June 2014, species identification was conducted via nested multiplex polymerase chain reaction and a reverse-hybridization assay of the internal transcribed spacer region (AdvanSure Mycobacteria GenoBlot Assay; LG Life Sciences, Seoul, South Korea) 27 , 28 . The radiological form of MAC-PD was categorized as FC, cavitary NB, or non-cavitary NB form 18 .…”

Mutation patterns of resistance genes for macrolides, aminoglycosides, and rifampicin in nontuberculous mycobacteria isolates from Kenya

“…Primers Size Reference rrl F (5 0 -TTTAAGCCCCAGTAAACGGC-3 0 ) R (5 0 -GTCCAGGTTGAGGGAACCTT-3 0 ) 420 bp (Park et al, 2020) rpoB F (5 0 -GGCAAGGTCACCCCGAAGGG-3 0 ) R (5 0 -AGCGGCTGCTGGGTGATCATC-3 0 ) 723 bp (Adékambi et al, 2003) rrs F (5 0 -AAGGAGGTGATCCAGCCGCA-3 0 ) R (5 0 -TCCCTTGTGGCCTGTGTGCA-3 0 ) 341 bp (Kim et al, 2021) The PCR products were enzymatically purified using ExoSAP IT (Applied Biosystems, Foster City, California, USA). Purification conditions were done at 37°C for fifteen minutes followed by a second incubation at 80°C for fifteenminutes and a final cooling step at 4°C for five minutes.…”

Mutation patterns of resistance genes for macrolides, aminoglycosides, and rifampicin in nontuberculous mycobacteria isolates from Kenya

“…341 bp (Kim et al, 2021) The PCR assays were carried out with a Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA) (Table 1). Thermal cycling conditions for rrl were as follows: one cycle of 95°C for five minutes, 35 cycles of 95°C for one minute, 55°C for one minute, 72°C for one minute, and a final extension for ten minutes at 72°C.…”