Spoligotyping and whole-genome sequencing analysis of lineage 1 strains of Mycobacterium tuberculosis in Da Nang, Vietnam

Abstract

BackgroundSpacer oligonucleotide typing (spoligotyping), a widely used, classical genotyping method for Mycobacterium tuberculosis complex (MTBC), is a PCR-based dot-blot hybridization technique to detect the genetic diversity of the direct repeat (DR) region. Of the seven major MTBC lineages in the world, lineage 1 (Indo-Oceanic) mostly corresponds to the East African–Indian (EAI) spoligotype family in East Africa and Southeast Asia.ObjectivesWe investigated the genomic features of Vietnamese lineage 1 strains, comparing spoligotype patterns using whole-genome sequencing (WGS) data.MethodsM. tuberculosis strains isolated in Da Nang, Vietnam were subjected to conventional spoligotyping, followed by WGS analysis using a high-throughput sequencer. Vietnamese lineage 1 strains were further analyzed with other lineage 1 strains obtained from a public database.ResultsIndicating a major spoligotype in Da Nang, 86 (46.2%) of the 186 isolates belonged to the EAI family or lineage 1. Although typical EAI4-VNM strains are characterized by the deletion of spacers 26 and 27, 65 (75.6%) showed ambiguous signals on spacer 26. De novo assembly of the entire DR region and in silico spoligotyping analysis suggested the absence of spacer 26, and direct sequencing revealed that the 17th spacer sequence not used for conventional typing, was cross-hybridized to the spacer 26 probe. Vietnamese EAI4-VNM, other EAI-like strains, and those showing a non-EAI pattern lacking many spacers formed a monophyletic group separate from other EAI families in the world.ConclusionInformation about the alignment of spacers in the entire DR region obtained from WGS data provides a clue for the determination of experimentally ambiguous spoligo patterns. WGS data also helped to analyze the hidden relationships between apparently distinct spoligo patterns.

Whole genome sequencing, analyses of drug resistance-conferring mutations, and correlation with transmission of Mycobacterium tuberculosis carrying katG-S315T in Hanoi, Vietnam

“…Briefly, after trimming and excluding severely contaminated samples, sequence reads were mapped to the H37Rv genome (AL123456.3) by using BWA-MEM 0.7.15 (https://github.com/lh3/bwa), followed by variant calling with the Genome analysis toolkit (GATK version 3.7) 70 . Only paired-end fastq files with average depth more than 25X were accepted for the analysis.…”

Phylogenomics Sheds Light on the population structure ofMycobacterium bovisfrom a Multi-Host Tuberculosis System

“…Similar findings have been reported by other works that register clustering agreement between spoligotyping and WGS (Hauer et al, 2019; Lasserre et al, 2018). For five strains, the spoligotyping profile obtained by the reverse-hybridization method in the wet lab is not the same as the profile determined in silico through the vSNP pipeline, a finding that has also been reported by others (Hijikata et al, 2017; Maeda et al, 2020). This mismatch might contribute to discrepancies in phylogenetic trees topology generated with classical genotyping methods and the SNP data.…”

Phenotypic and genotypic features of the Mycobacterium tuberculosis lineage 1 subgroup in central Vietnam

“…The EAI4_VNM spoligotype, which is characterized by the absence of spacers 26 and 27, 29 to 32, and 34 of 43 in the clustered regularly interspaced short palindromic repeat (CRISPR) locus of the Mtb genome 17 , belongs to a major L1 subgroup in Vietnam 18 . In our previous study 16 in Da Nang city, a central area of Vietnam, we showed that these strains form a clade that is discrete from other EAI strains in different geographical regions in Asia and Africa. Carrying only a few IS 6110 copies is also a characteristic of these Vietnamese strains 16 , 19 .…”

“…In our previous study 16 in Da Nang city, a central area of Vietnam, we showed that these strains form a clade that is discrete from other EAI strains in different geographical regions in Asia and Africa. Carrying only a few IS 6110 copies is also a characteristic of these Vietnamese strains 16 , 19 . For a comprehensive understanding of the genotypic and phenotypic characteristics of these strains, we used whole genome sequencing (WGS) to analyze the whole sample set of 181 Mtb strains with clinico-epidemiological data in Da Nang in central Vietnam.…”

“…Extending nearly 2000 km from the north through the central area to the south, different distributions of Mtb lineages in each of these areas of Vietnam have been found. Beijing genotype strains, a major sublineage of L2, are predominant in the northern and southern regions, whereas EAI strains in L1 are more frequently seen in the central area 14 – 16 . The EAI4_VNM spoligotype, which is characterized by the absence of spacers 26 and 27, 29 to 32, and 34 of 43 in the clustered regularly interspaced short palindromic repeat (CRISPR) locus of the Mtb genome 17 , belongs to a major L1 subgroup in Vietnam 18 .…”