Comparative genome analysis reveals high-level drug resistance markers in a clinical isolate of Mycobacterium fortuitum subsp . fortuitum MF GZ001

Abstract

Introduction: Infections caused by non-tuberculosis mycobacteria are significantly worsening across the globe. M. fortuitum complex is a rapidly growing pathogenic species that is of clinical relevance to both humans and animals. This pathogen has the potential to create adverse effects on human healthcare.

Methods: The MF GZ001 clinical strain was collected from the sputum of a 45-year-old male patient with a pulmonary infection. The morphological studies, comparative genomic analysis, and drug resistance profiles along with variants detection were performed in this study. In addition, comparative analysis of virulence genes led us to understand the pathogenicity of this organism.

Results: Bacterial growth kinetics and morphology confirmed that MF GZ001 is a rapidly growing species with a rough morphotype. The MF GZ001 contains 6413573 bp genome size with 66.18 % high G+C content. MF GZ001 possesses a larger genome than other related mycobacteria and included 6156 protein-coding genes. Molecular phylogenetic tree, collinearity, and comparative genomic analysis suggested that MF GZ001 is a novel member of the M. fortuitum complex. We carried out the drug resistance profile analysis and found single nucleotide polymorphism (SNP) mutations in key drug resistance genes such as rpoB, katG, AAC(2')-Ib, gyrA, gyrB, embB, pncA, blaF, thyA, embC, embR, and iniA. In addition, the MF GZ001strain contains mutations in iniA, iniC, pncA, and ribD which conferred resistance to isoniazid, ethambutol, pyrazinamide, and para-aminosalicylic acid respectively, which are not frequently observed in rapidly growing mycobacteria. A wide variety of predicted putative potential virulence genes were found in MF GZ001, most of which are shared with well-recognized mycobacterial species with high pathogenic profiles such as M. tuberculosis and M. abscessus.

Discussion: Our identified novel features of a pathogenic member of the M. fortuitum complex will provide the foundation for further investigation of mycobacterial pathogenicity and effective treatment.

Keywords: Mycobacterium fortuitum; comparative genomic analysis; drug resistance; morphology; pathogenesis.

Figure 1

Growth kinetics and colony surface architecture of Mycobacterium species. (A) Growth kinetics detection of MF GZ001, M. abscessus GZ002, and M. smegmatis C² 155. (B) Colony size and morphology determination. MF GZ001 and M. abscessus GZ002 were grown in Middlebrook 7H11 agar media supplemented with Tween 80 at 37°C. (C) Individual colony surface architecture was measured by OLYMPUS TH4-200.

Figure 2

Circular representation of MF GZ001 genome displayed with Circos (version 0.69). The circular plot has seven levels. From outside to inside, the first is the information of genome position, the second is GC content information, the third is positive strand genes (marked in red color), the fourth is negative strand genes (marked in green color), the fifth is positive strand ncRNA data (marked in blue color), the sixth is negative strand ncRNA data (marked in purple color) and the seventh shows long repeats (>100 bp).

Figure 3

Diagram depicting genomic comparisons obtained using Mauve software. The alignment display is organized into one horizontal “panel” per input genome sequence. Each genome’s panel contains the name of the genome sequence, a scale showing the sequence coordinates for that genome, and a single black horizontal center line. Colored block outlines appear above and possibly below the center line. Each of these block outlines surrounds a region of the genome sequence that is aligned to part of another genome and is presumably homologous and internally free from genomic rearrangement. Regions outside blocks lack detectable homology among the input genomes. Inside each block, Mauve draws a similar profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Areas that are completely white were not aligned and probably contain sequence elements specific to a particular genome. The height of the similarity profile is calculated to be inversely proportional to the average alignment column entropy over a region of the alignment.

Figure 4

Comparative genomes representation. (A) Pan-genome of M. fortuitum complex. The white circle in the middle of the flower displayed core gene clusters, and the petals represent the unique number of clusters of each Mycobacterium species. (B) MF GZ001 and other mycobacterial strains genome-based Venn diagrams. The circles of different colors in the Venn diagram represent different species, and the numbers in the figure represent the numbers of gene families unique or common to each strain. In the petal diagram, each petal represents a specie. The numbers on the petals represent the number of gene families unique to the species, and the white circle in the middle represents the number of gene families shared by all strains.

Figure 5

Drug resistance-related genes distribution in MF GZ001 strain.