Identification of M. abscessus subspecies
M. abscessus isolates identification was performed using 16S gene sequencing and the subsp. identification was done using sequencing of the hsp65 gene. PCR amplification was done in a 50 μL reaction volume consisting of 47.5 μL premix (1 μM forward primer, 1 μM reverse primer), 0.25 μL of Qiagen Taq DNA polymerase (Qiagen, Hilden, Germany), and 2.5 μL of template DNA (Table 1). Amplification was done using the Applied Biosystems Veriti® 96-Well Thermal Cycler (Thermo Fisher Scientific, Applied Biosystems, Grand Island, NY). Thermocycler conditions for hsp65 were 95°C for 5 min, 45 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min, followed by 72°C for 5 min. The PCR products were purified using PCRClean DX magnetic beads (Aline Biosciences, Woburn, MA). Sequencing was done using Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific, Applied Biosystems, Grand Island, NY). Sequences were assembled using Lasergene SeqMan Pro (DNAStar, Inc., Madison, WI) and sequence comparison was done using BioNumerics 7.6.2 software. Reference strains Mycobacterium abscessus subsp. abscessus ATCC 19977T, Mycobacterium abscessus subsp. bolletii CCUG 50184T, and Mycobacterium abscessus subsp. massiliense CCUG 48898T were used.
Antimicrobial susceptibility testing
Phenotypic susceptibility testing for clarithromycin was done using Sensititre™ RAPMYCO AST plates (Trek Diagnostics, Thermo Fisher Scientific, Oakwood Village, OH) according to the manufacturer instructions. Plates were examined for the drug MICs on day 3 (and day 5 if growth in positive control well was not adequate) of incubation then further incubation at 30°C was done if day 3 or 5 results showed sensitivity. Plates were examined for inducible resistance on day 7, 10 and 14 of incubation.
Sequencing of erm(41) and rrl genes
PCR amplification was done in a total reaction volume of 50 μL which consisted of 25 μL Amplitaq Gold® 360 Mastermix (Thermo Fisher Scientific, Applied Biosystems, Foster City, CA), 22.5 μL forward and reverse primers (final concentration of 1 μM each), and 2.5 μL of template DNA for both erm(41) and rrl genes. Primers ermF and ermR1 were used to amplify the erm(41) gene while primers 19F and 21R amplified the rrl gene (Table 1). Thermocycler conditions for amplifying erm(41) were 95°C for 7.5 min for the initial denaturation, followed by 35 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec followed by a final extension of 72°C for 10 min. Conditions were the same for rrl except with an annealing temperature of 55°C instead of 60°C. The PCR products were purified using PCRClean DX magnetic beads. Sequencing primers are shown in Table 1. Sequencing and analysis were done as described above for hsp65 gene.
Real-time PCR assay
A 96-well plate assay was designed based on a modified protocol from Shamira Shallom and colleagues (2015) . The probe, Absc-chel 16S, was used for identification, erm(41)_probe1 was used to detect full-length erm(41), SNPs on position 28 of erm(41) were detected using probes erm(41)T28 and erm(41)C28, SNPs on position 2058 of rrl were detected using probes 23S_A2058 and 23S_C2058 (Table 2). Probes for rrl, 23S_A2058 and 23S_C2058, initially consisted of LNA (locked nucleic acids) from the Shallom protocol, however, the rrl probes used in this experiment were modified. This change would see a lower melting temperature for these probes and therefore, the 5’ end of these probes were extended by 7 base pairs using Mabs5 23S rRNA (GenBank accession number EU980535.1) to ensure the same melting temperature of around 60°C. Probes were made to a working concentration of 2.5 μM in a mix with their corresponding primers (Table 2) which were diluted to 10 μM. Reactions were done in a total reaction volume of 25 μL consisting of 12.5 μL TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific, Life Technologies, Austin, TX.), probes final concentration of 250 nM and forward and reverse primers at final concentration of 1 μM each, and 3.75 μL of template DNA. Each sample was run in duplicates with positive control for all wild type and mutants i.e., 16S, full length erm(41), T28T in erm(41), T28C in erm(41), A2058A in rrl, and A2058C in rrl and negative control (no sample DNA). Real-time PCR was done using the Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Applied Biosystems, Grand Island, NY). Thermocycler conditions included an initial denaturation of 95°C for 3 min, 40 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec.