Determination of M. abscessus complex
All material samples suspected of mycobacterial contamination in the Juntendo university hospital were cultured in mycobacteria growth indicator tube (MGIT; Becton Dickinson, USA) broth and incubated at 37°C in the BACTEC MGIT 960 (Becton Dickinson, USA) instrument with ambient air. MGIT positive tubes were classiﬁed as M. abscessus based on the results of DNA–DNA hybridization (DDH) analysis (DDH Mycobacterium “Kyokuto” kit; Kyokuto Pharmaceutical Industrial, Japan) or matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Detected species were reconfirmed as three subspecies of M. abscessus complex by sequencing the 16S rRNA, rpoB, hsp65, and erm genes (20, 21). All strains of M. abscessus were cultured on BD trypticase soy agar II with 5% sheep blood (Blood agar; Nippon Becton-Dickinson and Company, Japan) at 35°C for approximately 4 to 6 days in an aerobic atmosphere. The study protocol was approved by the Ethics Committee of Juntendo University School of Medicine (no. 18-010 and 19-038).
MALDI-TOF MS analysis
MALDI-TOF MS analysis was performed based on previously described methods (22). Colonies of M. abscessus complex on blood agar were scratched with a needle, and particles on the needle surface were diluted in 50 μL 80% trifluoroacetic acid. After incubation for 15 minutes at room temperature, the solution was added to 150 μL distilled water and 200 μL 100% acetonitrile, followed by a centrifugation step (16,200 × g, 2 min). One microliter of the cleared supernatant containing the bacterial extract was transferred onto a MALDI target plate (Bruker Daltonik, Germany). We overlaid dried spots with MALDI matrix (10 mg/mL α-cyano-4-hydroxy-cinnamic acid [α-HCCA] in 50% acetonitrile:2.5% trifluoroacetic acid) (Bruker Daltonik, Germany). After drying of the matrix, we conducted MALDI-TOF MS analysis with a Microflex LT/SH benchtop mass spectrometer (Bruker Daltonik, Germany) equipped with a 60-Hz nitrogen laser. We had optimized parameter settings (ion source 1 [IS1], 20 kV; IS2, 18.2 kV; lens, 6.85 kV; detector gain, 2854 V; gating, none) for the mass range between 2,000 and 20,000 Da. We achieved spectra in the positive linear mode with the maximum laser frequency. An external standard (bacterial test standard [BTS]) (Bruker Daltonik, Germany) was applied for instrument calibration. Data evaluation was performed by visually comparing spectra to search for peak shifts using flexAnalysis 3.4 (Bruker Daltonik, Germany).
PCR amplification and DNA sequencing
DNA was extracted from cultured colonies using the DNeasy UltraClean Microbial Kit (QIAGEN, Germany), and PCR was conducted using Ex Taq DNA polymerase, hot-start version (Takara, Japan) according to the manufacturer’s instructions. The gene-specific primer pairs used for PCR analysis are listed in Table 4; these primers were used in previous studies (23, 24). The sequencing PCR products were purified with the BigDye XTerminator purification kit (Life Technologies, USA) and samples were loaded on the ABI Prism 3130 Genetic Analyzer (Thermo Fisher Scientific, USA). The DNA sequencing results were analyzed using a BLAST search to identify sequence similarity between samples and the three species of M. abscessus complex.
Antimicrobial susceptibility testing
Susceptibility testing was performed according to Clinical and Laboratory Standard Institute (CLSI) guideline M24-A2 (25). MIC determinations and synergy testing were performed by the checkerboard method using frozen broth microdilution plates (Eiken Chemical Co., Ltd., Japan). The ranges of antibiotic concentrations tested were as follows: amikacin (AMK) 0.25 to 64 μg/mL, clarithromycin (CLR) 0.06 to 64 μg/mL, imipenem (IPM) 4 to 32 μg/mL, and moxifloxacin (MXF) 1 to 32 μg/mL. MICs of each antimicrobial agent were determined by broth microdilution methods as recommended by the CLSI. The panels were prepared with a 96-channel dispenser and stored at -80°C until use. CLR were dispensed alone in the first row, and IPM, AMK, or MXF were dispensed in the first column. Each well was inoculated with a concentration of 1 × 105 colony-forming units (CFU)/mL. The MICs were determined after 3, 7, 14 days of incubation at 35°C. The MIC breakpoints, indicating susceptible, intermediate, and resistant strains, were interpreted according to the CLSI criteria for amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, moxifloxacin, trimethoprim/sulfamethoxazole, and tobramycin (Table 5) (25). The effect of each agent combined with clarithromycin was evaluated using FIC index analysis (13).
Categorical variables were compared using the chi-square test or Fisher's exact test. The evaluation of changes in MIC was performed using the Wilcoxon signed-rank test. Differences were considered significant at p <0.05. When the chi-square test results were statistically significant, adjusted residuals were calculated to determine which particular associations were significant. Adjusted residuals were significant at p < 0.05 level if they were less than −1.96 or more than 1.96, and were significant at p < 0.01 level if they were less than −2.58 or more than 2.58. All statistical analyses were performed using the SPSS software program (version 20, IBM Japan, Japan).