Pulmonary disease caused by nontuberculous mycobacteria (NTM) is increasing worldwide. Mycobacterium avium is the most clinically significant NTM species in humans and animals, and comprises four subspecies: M. avium subsp. avium (MAA), M. avium subsp. silvaticum (MAS), M. avium subsp. paratuberculosis (MAP), and M. avium subsp. hominissuis (MAH). To improve our understanding of the genetic landscape and diversity of M. avium and its role in disease, we performed a comparative genome analysis of 79 M. avium strains. Our analysis demonstrated that MAH is an open pan-genome species. Phylogenetic analysis based on single nucleotide variants showed that MAH had the highest degree of sequence variability among the subspecies, and MAH strains isolated in Japan and those isolated abroad possessed distinct phylogenetic features. Furthermore, MAP strains, MAS and MAA strains isolated from birds, and many MAH strains that cause the progression of pulmonary disease were grouped in each specific cluster. Comparative genome analysis revealed the presence of genetic elements specific to each lineage, which are thought to be acquired via horizontal gene transfer during the evolutionary process, and identified potential genetic determinants accounting for the pathogenic and host range characteristics of M. avium.
subsp. mainly causes disseminated infection in immunocompromised hosts, such as individuals with human immunodeficiency virus (HIV) infection, and pulmonary infection in immunocompetent hosts. However, many aspects of the different types of subsp. infection remain unclear. We examined the antibiotic susceptibilities and genotypes of subsp. isolates from different hosts by performing drug susceptibility testing using eight antibiotics (clarithromycin, rifampin, ethambutol, streptomycin, kanamycin, amikacin, ethionamide, and levofloxacin) and variable-number tandem-repeat (VNTR) typing analysis for 46 isolates from the sputa of HIV-negative patients with pulmonary subsp. disease without previous antibiotic treatment and 30 isolates from the blood of HIV-positive patients with disseminated subsp. disease. Interestingly, isolates from pulmonary subsp. disease patients were more resistant to seven of the eight drugs, with the exception being rifampin, than isolates from HIV-positive patients. Moreover, VNTR typing analysis showed that the strains examined in this study were roughly classified into three clusters, and the genetic distance from reference strain 104 for isolates from pulmonary subsp. disease patients was statistically significantly different from that for isolates from HIV-positive patients ( = 0.0018), suggesting that subsp. strains that cause pulmonary and disseminated disease have genetically distinct features. Significant differences in susceptibility to seven of the eight drugs, with the exception being ethambutol, were noted among the three clusters. Collectively, these results suggest that an association between the type of subsp. infection, drug susceptibility, and the VNTR genotype and the properties of subsp. strains associated with the development of pulmonary disease are involved in higher levels of antibiotic resistance.
Background Clarithromycin (CLR) is the key drug in multidrug therapy for Mycobacterium avium complex (MAC) diseases and the only drug for which drug susceptibility is correlated with a clinical response in these diseases. In the case of CLR-resistant MAC, a point mutation is present at either position 2058 or 2059 of the peptidyl transferase active center in the domain V region of 23S rRNA at the macrolide binding site. Using conventional investigation, we clarified the correlation between drug susceptibility testing and mutation of drug resistance genes. In this study, we adapted a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene in M. avium isolates (Figure 1). Furthermore, we evaluated the usefulness as point-of-care testing (POCT) technology using clinical isolates. Figure 1. The designs of CLR resistance A2058G mutant-type mismatch primers used for the ARMS-LAMP assay. a) A strand-displacing DNA polymerase extends the DNA from FIP while separating from the DNA chain. The primer F3 binds to its complementary region on the DNA to displace the newly synthesized DNA. An analogous reaction is performed by BIP and B3. α (α = A, wild type; G, A2058G) and β (β = A, wild type; C, A2058G) are indicated by the point mutation at position 2058 of the 23S rRNA gene. The bold area indicates the mismatched base C (cytosine). b) The synthesized DNA self-anneals because of the complementary region at both ends and forms ‘dumbbell’ structures. c) After repeated rounds, a complementary region on the same chain is amplified. Methods Primers for ARMS-LAMP were designed using PrimerExplorerV5 software based on the nucleotide sequence data for 23S rRNA in M. avium strain 104 (Figure 2). Using the minimum inhibitory concentration of CLR, drug susceptibility was determined for 18 clinical M. avium isolates. Of these, eight CLR-susceptible and 10 CLR-resistant strains were analyzed by sequencing the 23S rRNA gene and ARMS-LAMP. Figure 2. Alignment of the nucleotide sequences including the domain V region of 23S rRNA at the macrolide binding site. The constructed LAMP primer sets are shown in solid boxes (forward primers, F1-3) and dashed boxes (backward primers, B1-3). The bold area indicates the point mutation at position 2058 or 2059 of the 23S rRNA gene. Results Sequence analysis revealed that all eight CLR-sensitive strains tested were wild type, whereas all 10 CLR-resistant strains were mutants. Using ARMS-LAMP, no amplification with the mutant-type mismatch primer sets (MTPS) was observed in the eight wild-type strains, but amplification was observed with MTPS in the 10 mutant strains (Table 1). Table 1. MICs of CLR and results of ARMS–LAMP using Mycobacterium avium isolates. Conclusion The developed rapid detection method for the CLR resistance gene using ARMS-LAMP can determine drug resistance in a few hours without the need for special equipment. ARMS-LAMP may be a new clinically beneficial POCT technology for examination that is novel and extremely practical. Disclosures All Authors: No reported disclosures
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