Comprehensive subspecies identification of 175 nontuberculous mycobacteria species based on 7547 genomic profiles

Abstract

The prevalence of nontuberculous mycobacteria (NTM) pulmonary diseases has been increasing worldwide. NTM consist of approximately 200 species and distinguishing between them at the subspecies level is critical to treatment. In this study, we sequenced 63 NTM genomes, 27 of which were newly determined, by hybrid assembly using sequencers from Illumina and Oxford Nanopore Technologies (ONT). This analysis expanded the available genomic data to 175 NTM species and redefined their subgenus classification. We also developed a novel multi-locus sequence typing (MLST) database based on 184 genes from 7547 assemblies and an identification software, mlstverse, which can also be used for detecting other bacteria given a suitable MLST database. This method showed the highest sensitivity and specificity amongst conventional methods and demonstrated the capacity for rapid detection of NTM, 10 min of sequencing of the ONT MinION being sufficient. Application of this methodology could improve disease epidemiology and increase the cure rates of NTM diseases.

High Prevalence of Colistin-Resistant Escherichia coli with Chromosomally Carried mcr-1 in Healthy Residents in Vietnam

“…Of the 57 COR E. coli isolates with mcr, a total of 14 isolates were chosen for genome sequencing (Table 1). Whole-genome sequencing and assembly of isolates, along with a plasmid harboring mcr-1, were performed on the Illumina MiSeq (Illumina Inc., CA) and MinION (Oxford Nanopore Technologies, London, United Kingdom) sequencers, as described previously (22). The genomes were annotated using DDBJ Fast Annotation and Submission Tool pipeline (https://dfast.nig.ac.jp).…”

Complete Genome Sequence of Mycobacterium xenopi JCM15661 T , Obtained Using Nanopore and Illumina Sequencing Technologies

“…The ANI values with respect to two reported draft genomes of the strain (named DSM43995 and NCTC10042 in the NCBI database) were 99.89% (versus strain DSM43995) and 99.87% (versus strain NCTC10042). Also, the ANI values with respect to draft genomes of Mycobacterium heckeshornense (strain RLE) and Mycobacterium noviomagense (strain DSM45145), which are the mycobacterial species phylogenetically closest to M. xenopi (16)(17)(18), were 89.43% and 82.93%, respectively, confirming the taxonomic position of M. xenopi. The numbers of predicted coding sequences, rRNA operons, and tRNAs in the genome were 4,898, 6, and 47, respectively, nearly equivalent to those of the two previously reported draft genomes of M. xenopi.…”

Host Immune Response and Novel Diagnostic Approach to NTM Infections

“…However, the average nucleotide identity index cannot distinguish two genetically closely related but different species, notably, various concepts of species (e.g., biological, ecological, and genomic aspects) coexist in the taxonomy classification [15,94]. For mycobacterial identification, large-scale extensions of the MLST approach, such as PubMLST [95] or mlstverse [96], are used because of the requirement of subspecies level identification. PubMLST is a widely used MLST approach, consisting of conventional MLST and ribosomal MLST for bacterial identification.…”

“…From a clinical perspective, the mycobacterial incubation time is a bottleneck in the overall diagnostic process, because up to 8 weeks of growth may be needed to obtain culture isolates of slow-growing species. With the MLST approach using comprehensive genomic information regarding mycobacteria, the Oxford Nanopore Technologies MinION sequencer enables diagnosis within 20 min from isolated genomic DNA with mlstverse [96] (Figure 2). In addition, NGS technologies may allow direct detection of pathogens from metagenomic DNA because they present sequencing results in real-time.…”