Molecular Epidemiological Characteristics of Mycobacterium abscessus Complex Derived from Non-Cystic Fibrosis Patients in Japan and Taiwan

Abstract

Mycobacterium abscessus complex (MABC) is a group of emerging, highly antimicrobial-resistant non-tuberculous mycobacteria. Specific MABC clones are spreading globally in patients with cystic fibrosis (CF); however, associated genomic epidemiology is lacking in East Asia, with very few patients with CF. Here, we investigated MABC populations derived from non-CF patients in Japan and Taiwan. Analysis of whole-genome sequencing data of 220 MABC isolates revealed that 112, 105, and 3 were M. abscessus subsp. abscessus (ABS), M. abscessus subsp. massiliense (MAS), and M. abscessus subsp. bolletii (BOL), respectively. Moreover, >50% of ABS and >70% of MAS were related to four predominant clones in the region. Known mutations conferring macrolide resistance were rare (1.4%) and were not enriched in the predominant clones. Conversely, the macrolide-susceptible erm(41) T28C mutation was significantly enriched in one predominant ABS clone. The most predominant ABS clone was genetically related to the previously described dominant circulating clone (DCC)1 in patients with CF, whereas no isolates were related to DCC2; isolates related to DCC3 were not necessarily predominant in our sample set. We found that the erm(41) T28C mutants spread globally, and some of them reacquired the functional erm(41) gene through both point mutation and recombination. This study revealed predominant MABC clones in Japan and Taiwan and their relationship with the globally superadding clones in the patient community with CF. Our study provides insights into the genetic characteristics of globally dominant and area-specific strains isolated from patients with or without CF and differences between globally spread and regionally specific strains. IMPORTANCE Members of Mycobacterium abscessus complex (MABC) are frequently isolated from patients. Studies have reported that predominant clones of MABC (known as dominant circulating clones; DCCs) are distributed worldwide and transmitted from humans to humans in patients with cystic fibrosis (CF). However, associated genomic epidemiology has not yet been conducted in East Asia, including Japan and Taiwan, where there are only a few patients with CF. Using whole-genome sequencing data derived from non-CF patients in Japan and Taiwan, we revealed prevalent clones and the incidence of macrolide resistance-associated mutations in the MABC population in this region. We also clarified the associations between these predominant clones and DCCs in the global CF patient community. Our results would assist further studies in elucidating the genetic characteristics of strains isolated from patients with or without CF, the differences between globally spread and regionally specific strains, and the adaptive evolution of MABC within the host.

Keywords: Mycobacterium abscessus; molecular epidemiology; non-cystic fibrosis; non-tuberculous mycobacteria.

FIG 1

Phylogeny of 220 clinical isolates of MABC in Japan and Taiwan. Core-genome alignment of 220 isolates and 3 reference strains (ABS ATCC19977, MAS JCM15300, and BOL BD) of MABC was generated. A complete genome sequence of ATCC19977 was used as a reference. An alignment containing 235,540 recombination-free variable positions was used with RAxML to construct a maximum likelihood tree with 300 bootstrap replicates. Bootstrap values for the major nodes are shown. Scale bar indicates the mean number of nucleotide substitutions per site (SNPs/Site) on the respective branch. The red, green, and blue boxes indicate the location where each clinical isolate was obtained, and a gray box indicates the reference strain (ATCC19977). Pie charts indicate the ratio of the three subspecies of isolates identified in all (n = 220), Tokyo (Japan, n = 92), Okinawa (Japan, n = 25), and Taipei (Taiwan, n = 103), respectively.

FIG 2

Clustering analysis of ABS in Japan and Taiwan and mutations associated with inducible or acquired macrolide resistance. A core-genome alignment of 112 ABS clinical isolates and a reference strain ATCC19977 was generated (=3,963,788 bp, covering 78.2% of the reference genome). The alignment containing 76,114 recombination-free variable positions within the core genome was used to construct a maximum likelihood tree with 300 bootstrap replicates. Bootstrap values > 98% for the major nodes are shown. Six monophyletic clusters (ABS-EA1 to ABS-EA6) identified using TreeGubbins are shown. The pie chart indicates the proportion of the identified clusters, and the two dominant clusters (ABS-EA1 and ABS-EA2) are depicted in red and green, respectively. The location where each clinical isolate was isolated is indicated, as shown in Fig. 1. The presence (black) and absence (white) of macrolide resistance-associated mutations are indicated. Scale bar; the mean number of nucleotide substitutions per site (SNPs/Site) on the respective branch.

FIG 3

Clustering analysis of MAS in Japan and Taiwan and mutations associated with inducible or acquired macrolide resistance. A core-genome alignment of 105 MAS clinical isolates and a reference strain JCM15300 was generated (=4,033,769 bp, covering 81.0% of the reference genome). The alignment containing 48,718 recombination-free variable positions located within the core-genome was used to construct a maximum likelihood tree with 300 bootstrap replicates. Bootstrap values > 98% for the major nodes are shown. Five monophyletic clusters (MAS-EA1 to MAS-EA5) identified using TreeGubbins are shown. The pie chart indicates the proportion of identified clusters, and the two dominant clusters (MAS-EA1 and MAS-EA2) are depicted in blue and orange, respectively. The location where each clinical isolate was isolated and the presence (black) and absence (white) of macrolide resistanceassociated mutations are the same as those in Fig. 2. Scale bar; the mean number of nucleotide substitutions per site (SNPs/Site) on the respective branch.

FIG 4

Phylogenetical association between ABS isolates in Japan and Taiwan and those in other countries. Phylogeny of ABS from several countries was estimated using a core-genome alignment of 461 clinical isolates from four regions (Australia, East Asia, Europe, and North America). The complete genome sequence of ATCC19977 was used as a reference. The lignment containing 102,613 recombination-free variable positions in the core genome (3,584,621 bp, covering 70.7% of the reference genome) was used with RAxML to construct a maximum likelihood tree with 300 bootstrap replicates. The 16 monophyletic clusters (ABSGL1 to ABS-GL16) identified using TreeGubbins, and the corresponding clusters identified in Fig. 2, and Bryant et al. (2016) are shown. The presence (black) and absence (white) of inducible macrolide resistance-associated mutations in the erm(41) gene are indicated. Disease status (CF; yellow and non-CF; white) of the corresponding patients are shown. Each color box corresponds to the region where the clinical isolate was isolated. Asterisks indicate clusters that consist of isolates from more than two regions. Scale bar indicates the mean number of nucleotide substitutions per site (SNPs/Site) on the respective branch.

FIG 5

Reacquisition of wild-type T28 genotype in erm(41) of ABS clinical isolates belonged to ABS-GL3. (A) Recombination events in the genomic region around the erm(41) gene of ABS clinical isolates belonged to the ABS-GL3 cluster. Red or blue shaded boxes indicate common or sporadic recombination events. The presence and absence of mutations in the erm(41) gene, disease status (CF or non-CF) of corresponding patients, and the region where the clinical isolate was isolated are shown as Fig. 4. (B) A schematic depiction regarding the reversion of the wild-type erm(41) T28 genotype. Black and magenta arrows indicate genes, the fourth of which is erm(41).

FIG 6

Phylogenetical association between MAS in Japan and Taiwan and those in other countries. Phylogeny of global MAS was estimated using core-genome alignment of 233 clinical isolates from five regions (Australia, East Asia, Europe, North America, and South America). The complete genome sequence of JCM15300 was used as a reference. The alignment containing 58,975 recombination-free variable positions located in the core genome (3,789,583, covering 76.1% of the reference genome) was used with RAxML to construct a maximum likelihood tree with 300 bootstrap replicates. The 11 monophyletic clusters (MASGL1 to MAS-GL11) identified using TreeGubbins, and the corresponding clusters identified in Fig. 3, and Bryant et al. (2016) are shown. In Fig. 4, the presence and absence of mutations in the erm(41) gene, disease status (CF or non-CF) of the corresponding patients, and the region where the clinical isolate was isolated are shown. Asterisks indicate clusters that consist of isolates from more than two regions. Magenta boxes indicate clinical isolates that caused nosocomial outbreaks of MAS (15, 32). Scale bar indicates the mean number of nucleotide substitutions per site (SNPs/Site) on the respective branch.

FIG 7

Genetic components associated with MAS isolates that were genetically related to previously reported outbreak strains. (A) Sub-cluster analysis of MAS belonged to the MASGL1 cluster. The phylogenetic tree of MAS clinical isolates was constructed as described in Fig. 6. Sub-clusters were inferred using fastBAPS (47) with default settings, in which the coregenome alignment of clinical isolates belonging to MAS-GL1 was used as an input file. The presence (black) and absence (white) of the dpnIIA gene, disease status (CF or non-CF) of the corresponding patients, and the region where the clinical isolate was isolated are shown. (B) Examples of acquisition of the dpnIIA locus between clinical isolates belonged to MAS-GL1.2 and MAS-GL1.3. Arrows indicate genes annotated with DFAST-core (48), and dotted arrows indicate pseudogenes estimated by DFAST-core. Orthologous genes between clinical isolates are shown with red connections and are plotted with genoPlotR (49). Five genes exclusively associated with clinical isolates that belonged to MAS-GL1.2 and MAS-GL1.3 are colored in yellow.