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. 2025 Oct 15:16:1579154.
doi: 10.3389/fmicb.2025.1579154. eCollection 2025.

Virulence and mutations analysis based on the whole genome of a Brazilian Corynebacterium diphtheriae strain isolated from a cutaneous infection

Max Roberto Batista Araújo #  1   2 , Louisy Sanches Dos Santos #  3 , Fernanda Diniz Prates  1   2 , Hugo Felix Perini  4 , Jailan Sousa Silva  2 , Juliana Nunes Ramos  3 , Sérgio Bokermann  5 , Cláudio Tavares Sacchi  6 , Ana Luíza de Mattos Guaraldi  3 , Karoline Rodrigues Campos  6 , Tayná do Carmo Sant'Anna Cardoso  3 , Diogo Luiz de Carvalho Castro  2 , Marcos Andrade Silva  3 , Mireille Ângela Bernardes Sousa  1 , Verônica Viana Vieira  7 , Marlon Benedito Nascimento Santos  6 , Carlos Henrique Camargo  5 , Bruno Silva Andrade  8 , Marcos Vinicius da Silva  4 , Lincoln de Oliveira Sant'Anna  3 , Marcus Vinícius Canário Viana  2 , Vasco Azevedo  2
Affiliations

Virulence and mutations analysis based on the whole genome of a Brazilian Corynebacterium diphtheriae strain isolated from a cutaneous infection

Max Roberto Batista Araújo et al. Front Microbiol. .

Abstract

Corynebacterium diphtheriae is the main etiological agent of diphtheria, a potentially fatal disease whose most severe signs and symptoms result from the action of an exotoxin, the diphtheria toxin (DT). Although non-toxigenic C. diphtheriae strains have been associated with several diseases, including cutaneous infections and endocarditis, they are not monitored in many countries, and their mechanisms of virulence and antimicrobial resistance remain underexplored. Therefore, this study aimed to provide a comprehensive characterization -through genomic, in vitro, and in vivo analyses - of a non-toxigenic C. diphtheriae strain (46855) isolated from a leg lesion, highlighting its pathogenic potential and resistance profile. The isolate was assigned to a novel sequence type (ST-925) and was found to be resistant to tetracycline and rifampin. Multiple antimicrobial resistance genes were predicted in the genome, such as tet(33), rbpA, and rpoB2, in addition to mutations in the rpoB gene. A diverse set of virulence-associated genes related to adhesion, iron uptake systems, gene regulation, and post-translational modification was also identified. The isolate was able to form biofilm in vitro and exhibited strong virulence in Galleria mellonella larvae and A549 human pneumocyte cells. Finally, the structural analysis of the rpoB gene, carried out for the first time in this study, linked the observed mutations to rifampin resistance in C. diphtheriae. In summary, the data revealed that C. diphtheriae 46855, although non-toxigenic, harbors multiple genes associated with antimicrobial resistance and virulence, emphasizing the need for greater surveillance and functional studies on non-toxigenic strains.

Keywords: CRISPR-Cas system; Corynebacterium diphtheriae complex; non-toxigenic; resistance genes; virulence factors.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Phylogenetic tree showing relationships among various strains of *Corynebacterium diphtheriae* and *Lawsonella clevelandensis*. The tree includes different strains labeled with identification numbers. Bootstrap values are indicated on the branches, with key values highlighted, such as 0.852 and 0.99.
FIGURE 1
Phylogenetic tree genome shows the bootstrap percentage with 1000 bootstraps on the tree branches, ranging from 0.951 as the lowest percentage to 1 as the highest one. The outgroup was represented by Lawsonella clevelandensis X1036.
“Molecular structure of the rifampin–Mycobacterium tuberculosis RNA polymerase β-subunit complex (5UHC_B). (A) Overall view of the β-subunit bound to Rifampin, showing the crystallized position (orange) and the redocked pose (blue). (B) Close-up of Rifampin’s binding pocket, illustrating interactions with key amino acid residues. Bond types are indicated as follows: conventional hydrogen bonds (green), carbon–hydrogen bonds (light green), unfavorable positive–positive interactions (red), alkyl interactions (pink), and π–alkyl interactions (light pink).”
FIGURE 2
5UH_B redocking with rifampin. (A) Mycobacterium tuberculosis beta subunit with RNA and rifampin native (orange) and redocking (blue). (B) 2D interaction map between rifampin and 5UHC_B binding site residues.
“Docking analysis of rifampin with mutant Corynebacterium diphtheriae RpoB (rpoB-N441Y-S445F). (A) Overall structure of the RpoB–rifampin complex, with rifampin depicted in orange within its binding pocket. (B) Close-up of rifampin’s interactions with key amino acid residues, including Gln-433, Arg-463, Phe-437, Tyr-441, and Pro-487.”
FIGURE 3
Docking experiment Corynebacterium diphtheriae rpoB and rifampin. (A) Beta subunit complexed with rifampin in native position (orange) and docked position (blue). (B) 2D interaction map between rifampin and rpoB-N441Y-S445F binding site residues.
“Docking analysis of rifampin with wild-type and mutant Corynebacterium diphtheriae RpoB. (A) Rifampin bound to the wild-type RpoB (rpoB-N441-S445), showing interactions with key amino acid residues in the binding pocket. (B) Superposition of the wild-type (rpoB-N441-S445, blue) and mutant (rpoB-N441Y-S445F, orange) binding pockets within 5 Å of the rifampin site, highlighting amino acid substitutions between the two proteins.”
FIGURE 4
Corynebacterium diphtheriae rpoB-N441-S445 Binding site residues. (A) 2D interaction map between rifampin. (B) rpoB-N441-S445 (blue) and rpoB-N441Y-S445F (Orange) Superposition in rifampicin binding site at 5 angstroms contact, highlighting amino acid substitutions.
Panel A shows a bar graph depicting optical density at 600 nm over four time points: 1, 3, 6, and 24 hours, with increasing values and significant differences indicated by asterisks. Panels B to E display micrographs showing a progressive increase in cell density from sparse to dense clusters, corresponding to the time points in the graph.
FIGURE 5
Biofilm biomass formation of Corynebacterium diphtheriae 46855 by crystal violet method (A); representative biomass formation after 1 h (B), 3 h (C), 6 h (D) and 24 h (E). ANOVA with Tukey’s post-test. **p < 0.01, ****p < 0.001. Scale bar = 200 μm.
“Kaplan‑Meier survival curve showing survival over time for different treatment groups. The x-axis shows time in hours; the y-axis shows survival percentage. Treatment doses ranged from 103 to 1010 (each group represented by a different symbol). Statistically significant differences between groups are marked with asterisks.”
FIGURE 6
Keplan-Meier’s survival curve of Galleria mellonella larvae infected with 1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107, 1 × 108, 1 × 109, and 1 × 1010 cells/mL of Corynebacterium diphtheriae 46855. Data showed that the 1×109 and 1×1010 cells/mL inoculum resulted in significantly higher mortality than less concentrated inoculum and control condition (PBS). Logrank test *p < 0.05; ****p < 0.001.
Three bar graphs labeled A, B, and C depict CFU count over time post-infection at 1, 2, 3, 4, and 6 hours. Each color represents different datasets: A (teal), B (purple), and C (pink). Significant differences between bars are noted with p-values, all less than 0.001. Bars increase and decrease at varying rates across graphs.
FIGURE 7
Number of viable bacteria of Corynebacterium diphtheriae 46855 strain associated with (A), internalized by (B) and in the supernatant of (C) A549 monolayers in different periods. Data presented as the mean ± standard deviation of three independent experiments carried out in 4 replicates. Data were considered statistically different when p ≤ 0.05. CFU, colony forming units.
Three-panel image comparing cell samples labeled A, B, and C. Panel A shows several cells with prominent purple-stained nuclei. Panel B shows cells with black granular deposits surrounding the purple nuclei. Panel C displays a similar pattern to Panel B, with scattered purple-stained nuclei and granular deposits.
FIGURE 8
Micrographs of A549 cells infected with the Corynebacterium diphtheriae 46855 strain. (A) Non-infected control; (B) 3 h post-infection; (C) 6 h post-infection. Magnification ×400. Scale bar equal to 35 μm.
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