In this study, the MABSC clinical isolates were identified to the subspecies level, and their susceptibilities to 16 antimicrobial agents were determined. In addition, the clinical characteristics of the patients were investigated. In Thailand, this information is still limited, as only a few studies have reported the prevalence and drug susceptibility profiles of MABSC [7, 23]. MABSC is the most common RGM isolated from clinical specimens at our hospital. True MABSC pulmonary infections or NTM-PD can be diagnosed using clinical, radiological, and microbiological criteria [16, 17] and differentiated from specimen contamination [24] or colonization in the respiratory tract. For the diagnosis of extrapulmonary NTM disease, a single positive specimen from a sterile site, body fluid, or tissue biopsy is sufficient [25]. These criteria were applied for the diagnosis of MABSC infection in this study. Most patients with MABSC infections or colonization at our hospital had comorbidities such as immunocompromised status and bronchiectasis, which are associated with NTM infections [13, 26]. In addition, the rate of infections related to medical procedures has increased.

To treat NTM-PD infections, a combination of antimicrobial drugs including oral (azithromycin or clarithromycin, clofazimine, linezolid) and intravenous drugs (amikacin, imipenem or cefoxitin, tigecycline), is recommended and applied for extrapulmonary diseases. However, the optimal duration of treatment has not been universally established [16, 17]. Drug susceptibilities should be determined for clinically significant MABSC isolates. However, MABSC drug susceptibilities may not be the only key factor that influences the clinical outcomes of infected patients. In this study, some patients with favorable outcomes were infected by ICR-MABSC but responded to macrolide-based regimens. However, certain patients who were infected with clarithromycin/amikacin-susceptible MABSC strains experienced treatment failure or death (Table S2). Therefore, other factors, such as drugs used in the regimen, duration of use, severity of the disease, or patient comorbidities [27], could impact the clinical outcomes for the individual patient. In this study, the MABSC subspecies were not statistically associated with clinical outcomes. This could be due to a low number of patients (n = 31) from loss to follow-up, one of the limitations of this study.

The major MABSC subspecies that caused infections and were recovered from clinical specimens in our hospital were MAB followed by MMA. MBO was a relatively rare pathogen, consistent with findings in other countries. In addition, the drug susceptibility patterns of each MABSC subspecies and clinical isolates from various regions and countries can be different [5,6,7,8,9]. Therefore, the MABSC subspecies could be one of the important factors in the selection of an optimal and effective therapeutic regimen for patient management. In this study, MAB was associated with clarithromycin and amikacin resistance. However, the subspeciation analysis of MABSC is not available in most routine diagnostic laboratories. Therefore, the drug susceptibility patterns of the clinical isolates of MABSC without subspeciation are shown in Table 3. Amikacin was still the most effective drug against MABSC, and most MABSC strains were highly resistant to several drugs, including clarithromycin (day 14 of incubation), ciprofloxacin, doxycycline, moxifloxacin, TMP/SMX, and tobramycin, as previously reported [5, 6, 9]. A high intermediate rate was observed for imipenem and cefoxitin, consistent with the findings from previous studies [8, 9, 28]. For drugs without addressed breakpoints, clofazimine and tigecycline could be potential drugs to effectively treat MABSC infections due to their low MIC50 and MIC90 values, consistent with a study from the United States [8]. On the other hand, amoxicillin/clavulanic acid, cefepime, ceftriaxone, and minocycline may be ineffective in inhibiting the pathogens, so they have not been recommended for MABSC treatment [16, 17].

At present, molecular testing has become an important tool for rapidly detecting the gene mutations associated with clarithromycin and amikacin resistance. In this study, discrepant results between phenotypic and genotypic (GenoType NTM-DR) drug susceptibility results were observed, as previously reported [7, 29]. This indicated that clarithromycin and amikacin resistance might involve other mechanisms apart from those mutations detected in the erm(41), rrl, and rrs genes. In addition, despite the presence of T28, three MAB isolates did not express ICR. These findings suggest a point mutation, especially nonsynonymous or nonsense mutation, leading to a truncated Erm protein [30]. Therefore, sequencing analysis of erm(41) should be performed.

For colony morphology, MABSC can switch from a smooth to a rough morphotype, which has been shown to be more invasive and associated with poor clinical outcomes [13, 14]. Our findings show that the rough morphotype was significantly associated with amikacin, clarithromycin, and imipenem resistance, while the smooth morphotype was associated with linezolid resistance. However, the correlations between MABSC morphotype and drug susceptibility are still limited and lacking clarity. A French study reported high MICs of imipenem and cefoxitin in the rough morphotype [31], which was consistent with our findings for imipenem. However, previous studies showed that the MABSC morphotype did not significantly impact antimicrobial susceptibility [32, 33]. Therefore, future genetic analysis of glycolipid (GLP) synthesis or transport genes [34] of MABSC clinical isolates should be performed to confirm their true morphotypes and should study their correlation with drug susceptibility or clinical outcomes.

This study has many strengths, as it is the first study to investigate the clinical and microbiological associations of patients with MABSC infections in Thailand. However, there are some limitations in this study. First, this study included MABSC isolates from both treated and nontreated patients, which could affect the drug susceptibilities from the selection of resistant strains in treated patients. Second, the number of patients and MBO isolates was low. Third, the discrepant results between the phenotypic and genotypic susceptibilities of clarithromycin and amikacin were not further investigated using other methods, such as gene sequencing analysis. Last, this was a single-center study, and most patients came from Central Thailand. None of the patients living with HIV who were particularly vulnerable to NTM infection were recruited for this study. Therefore, these limitations could impact the statistical analysis and might not represent all MABSC isolates from the Thai population and patients living with HIV. Future studies will be conducted with additional patients and MABSC isolates, as well as genetic analysis of the genes associated with MABSC morphotypes and drug susceptibility.