The Use of Comparative Genomic Analysis for the Development of Subspecies-Specific PCR Assays for Mycobacterium abscessus

Abstract

Mycobacterium abscessus complex (MABC) is an important pathogen of immunocompromised patients. Accurate and rapid determination of MABC at the subspecies level is vital for optimal antibiotic therapy. Here we have used comparative genomics to design MABC subspecies-specific PCR assays. Analysis of single nucleotide polymorphisms and core genome multilocus sequence typing showed clustering of genomes into three distinct clusters representing the MABC subspecies M. abscessus, M. bolletii and M. massiliense. Pangenome analysis of 318 MABC genomes from the three subspecies allowed for the identification of 15 MABC subspecies-specific genes. In silico testing of primer sets against 1,663 publicly available MABC genomes and 66 other closely related Mycobacterium genomes showed that all assays had >97% sensitivity and >98% specificity. Subsequent experimental validation of two subspecies-specific genes each showed the PCR assays worked well in individual and multiplex format with no false-positivity with 5 other mycobacteria of clinical importance. In conclusion, we have developed a rapid, accurate, multiplex PCR-assay for discriminating MABC subspecies that could improve their detection, diagnosis and inform correct treatment choice.

Identification of Leuconostoc species based on novel marker genes identified using real-time PCR via computational pangenome analysis

“…gelidum. Therefore, a comparison of core and pan-based trees showed differences in order within species cluster but no differences in species assignment, which is consistent with previous study (Akwani et al, 2022).…”

“…Consistently, previous studies reported that incorrectly assigned taxonomic labels for bacterial species are prevalent with reported (Ghosh et al, 2019;Yang et al, 2021;Akwani et al, 2022). For example, Lacticaseibacillus paracasei was misclassified as Lacticaseibacillus casei and Enterococcus lactis as Enterococcus faecium, all of which are closely related species (Ghosh et al, 2019;Kim et al, 2022a).…”

A multiplex PCR assay for the differentiation of Mycobacterium tuberculosis complex reveals high rates of mixed-lineage tuberculosis infections among patients in Ghana

“… An overview of Scheme of work towards identification of lineage-specific genes for PCR primers development (similar to comparative genomic workflow previously described by Akwani et al., 2022 ). …”

“…The reaction mix contained an excess of primers and nucleotides to ensure reaction continuity without limitation. The amplification An overview of Scheme of work towards identification of lineagespecific genes for PCR primers development (similar to comparative genomic workflow previously described by Akwani et al, 2022).…”

“…Using the comparative genomics workflow previously described by Akwani et al., 2022 , MTBC lineage-specific genes identified were transferred into the development of multiplex PCR assay for TB lineage differentiation. This will enhance precise disease diagnosis, improve epidemiological surveillance studies and help inform selection of appropriate TB drug regimens at early time point especially in low resource settings with high TB incidence.…”