Detection of a streptogramin A O-acetyltransferase gene (vatD) in the chromosome of Clostridium botulinum isolated from infants in the United States

Abstract

Infant botulism, the most common form of botulism in the United States, occurs when botulinum toxin-producing clostridia colonize the infant's gut and produce botulinum neurotoxin (BoNT). This condition is treated with botulinum antitoxin and supportive care; antibiotics are avoided due to the risk of worsening symptoms by promoting toxin release. Consequently, antimicrobial resistance genes (ARGs) are rarely investigated in Clostridium botulinum strains. During 2020 through 2023, 226 C. botulinum isolates associated with infant botulism were sequenced in our laboratory using whole-genome sequencing (WGS) technologies. In silico analysis of the WGS data revealed that 15% of these isolates (n = 36) carried a vatD gene variant, which is associated with resistance to streptogramin A antibiotics. In this study, we describe the molecular characterization of 36 isolates that harbor the vatD gene. To the best of our knowledge, this is the first report of the detection of the vatD gene in the chromosome of C. botulinum recovered from infants in the United States.IMPORTANCEThe continuous expansion of whole-genome sequencing (WGS) technologies, combined with refined data analysis tools, has enabled an in-depth analysis of pathogens, allowing a thorough characterization and comprehension of the genomic diversity of C. botulinum. In the present study, we demonstrate how the implementation of WGS into laboratory surveillance workflow allowed the detection of an antimicrobial resistance gene, the vatD gene, in the chromosome of C. botulinum strains recovered from infants. To the best of our knowledge, this is the first study to report the detection of the vatD gene in non-enterococci isolates recovered from clinical samples in the United States.

Keywords: Clostridium botulinum; antimicrobial resistance genes; bont gene; infant botulism; streptogramin A.

Fig 1

BRIG (Blast Ring Image Generator) comparison of bont/B1 carrying plasmid vs. closest putative matche. Sequence homology among four strains and pBT-22100019 (GenBank accession number: CP121697.1). Each ring corresponds to a putative plasmid sequence with the inner ring representing the reference. The color gradient indicates a BLAST result with a matched degree of identity. The bontB gene cluster is shown in black.

Fig 2

Multiple sequence alignment of the vatD amino acid sequences of two representative strains from our study and the reference (GenBank access number: NG_048540.1/WP_002295712). The different shades of purple represent the corresponding amino acid’s similarity (darkest purple tone = 100% similarity). The amino acids highlighted in red are key active site residues, fundamental to the catalytic activity of the vatD (Tyr-37, His-82, and Trp-121). Image generated using Jalview v. 2.11.4.1.

Fig 3

Comparative vatD gene cluster analysis among E. faecium BM4145 and a subset of C. botulinum strains harboring the vatD gene (pink arrow). Each gene is represented by a color, the direction of the arrows indicates the gene transcription direction, and the sequence identity of linked regions is represented by the shaded gradient. Global alignment and image generation were performed using clinker & clustermap.js v.0.0.25.

Fig 4

Maximum-likelihood tree based on the amino acid sequences of the vatD gene from 55 Clostridium strains and the reference, vatD_En (GenBank access number: NG_048540.1) as an outgroup. The number at each node represents the bootstrap values. The colored outer ring denotes the country of origin for each strain, and the clusters are indicated by the colored range labels. The tree was generated using MEGA X and visualized with iTOL v6.9.

Fig 5

Core genome SNP phylogenetic tree generated using IQ-Tree v.2.2.0.3 and visualized using iTOLv.6.9. SNP analysis was conducted using Snippy against the Okra B1 reference (accession: NC_010516.1). The inner color strip represents the corresponding US states, while the outer ring denotes the corresponding ST. The presence of the bont/B1 gene is denoted by a green square at the tips of the tree.

Fig 6

Visualization of the pangenome of vatD+ Clostridium strains. Pangenome analysis was performed using Panaroo. (A) Pangenome maximum likelihood tree compared to a matrix with the presence (blue) and absence (white) of core and accessory genes, purple represents the strains from our study (US CDC strains), yellow the strains from China, and light green the strain from Argentina. (B) Pie chart with the breakdown of the number of genes in the pangenome. (C) Pan-genome (light purple/red) versus core genome (yellow plot/green) plot to indicate the openness of the C. botulinum pangenome. The pangenome trajectory increases as more genomes are added to the analysis.