Mycobacterium abscessus is an important infectious agent highly associated with drug resistance and treatment failure. We investigated the drug resistance situation of M. abscessus in Northeast Thailand and the possible genetic basis for this. Sixty-eight M. abscessus clinical isolates were obtained from 26 patients at Srinagarind Hospital during 2012–2016. Drug susceptibility tests and sequencing of erm(41), rrl and rrs genes were performed. Mycobacterium abscessus was resistant to 11/15 antibiotics (nearly 100% resistance in each case). Partial susceptibility to four antibiotics was found (amikacin, tigecycline, clarithromycin and linezolid). Non-massiliense subspecies were significantly associated with clarithromycin resistance (p<0.0001) whereas massiliense subspecies were associated with tigecycline resistance (p = 0.028). Inducible clarithromycin resistance was seen in 22/68 (32.35%) isolates: 21 of these isolates (95.45%) belonged to non-massiliense subspecies and resistance was explicable by the T28C mutation in erm(41). Inducible clarithromycin resistance was found in one isolate of the massiliense subspecies. Acquired clarithromycin resistance explicable by the A2271G/C mutation of rrl was seen in only 7/16 (43.75%) of strains. Inducible and acquired resistance mechanisms can be interchangeable during the course of infection. Rrs mutations were not associated with amikacin resistance in our study. Antibiotic resistance in subspecies of M. abscessus was reported from Northeast Thailand. Known resistance-associated mutations cannot explain all of the resistance patterns observed.

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BackgroundNontuberculous mycobacterial (NTM) infection is increasing worldwide. Current epidemiological data and knowledge of risk factors for this disease are limited. We investigated the trends in and risk of NTM infection in Northeast Thailand during 2012–2016.MethodsPatient demographics, infection site(s), and underlying disease or conditions from 530 suspected cases of NTM infections were retrieved from medical records, reviewed and analyzed. A diagnosis of true NTM infection was accepted in 150 cases. Risk factor analyses were done for extrapulmonary NTM infections compared to pulmonary NTM infections and for Mycobacterium abscessus compared to members of the Mycobacterium avium complex (MAC). Trend analysis among NTM species causing NTM infections was performed.ResultsThe most common species of NTMs causing extrapulmonary (n = 114) and pulmonary (n = 36) NTM infections in Northeast Thailand were M. abscessus (25.4% of extrapulmonary infected cases and 27.8% of pulmonary cases) followed by MAC (14.9% of extrapulmonary and 13.9% of pulmonary cases). Presence of anti-IFN-γ autoantibodies was the major risk factor for extrapulmonary (odds ratio (OR) = 20.75, 95%CI [2.70–159.24]) compared to pulmonary NTM infection. M. abscessus infection was less likely (OR = 0.17; 95%CI [0.04–0.80]) to be found in patients with HIV infection than was MAC infection. The prevalence of NTM infection, especially M. abscessus, in Northeast Thailand has recently increased. Extrapulmonary NTM and complicated NTM infections have increased in concordance with the recent trend of increasing frequency of anti-IFN-γ autoantibodies in the population.ConclusionsM. abscessus was the commonest NTM pathogen followed by MAC. The prevalence of NTM infections and anti-IFN-γ are showing an upward trend. Autoimmune disease due to anti-IFN-γ is the major risk factor for extrapulmonary NTM infection in Northeast Thailand.

Mixed infection with multiple species of nontuberculous mycobacteria (NTM) is difficult to identify and to treat. Current conventional molecular-based methods for identifying mixed infections are limited due to low specificity. Here, we evaluated the utility of whole-genome sequencing (WGS) analysis to detect and identify mixed NTM infections. Analytical tools used included PubMLST, MetaPhlAn3, Kraken2, Mykrobe-Predictor and analysis of heterozygous SNP frequencies. The ability of each to identify mixed infections of NTM species was compared. Sensitivity was tested using 101 samples (sequence sets) including 100 in-silico simulated mixed samples with various proportions of known NTM species and one sample of known mixed NTM species from a public database. Single-species NTM control samples (155 WGS samples from public databases and 15 samples from simulated reads) were tested for specificity. Kraken2 exhibited 100% sensitivity and 98.23% specificity for detection and identification of mixed NTM species with accurate estimation of relative abundance of each species in the mixture. PubMLST (99% and 96.47%) and MetaPhlAn3 (95.04% and 83.52%) had slightly lower sensitivity and specificity. Mykrobe-Predictor had the lowest sensitivity (57.42%). Analysis of read frequencies supporting single nucleotide polymorphisms (SNPs) could not detect mixed NTM samples. Clinical NTM samples (n = 16), suspected on the basis of a 16S–23S rRNA gene sequence-based line-probe assay (LPA) to contain more than one NTM species, were investigated using WGS-analysis tools. This identified only a small proportion (37.5%, 6/16 samples) of the samples as mixed infections and exhibited only partial agreement with LPA results. LPAs seem to be inadequate for detecting mixed NTM species infection. This study demonstrated that WGS-analysis tools can be used for diagnosis of mixed infections with different species of NTM.

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