Comparative genomic analysis of food-animal-derived and human-derived Clostridium perfringens isolates from markets in Shandong, China

Abstract

Clostridium perfringens (C. perfringens) is a foodborne pathogen that poses a significant threat to both animal husbandry and public health. In this study, 27 C. perfringens strains were isolated from animal samples and animal-derived food products. Antibiotics resistances among the isolates were phenotypically and genotypically analyzed and Whole genome sequencing (WGS). In combination with the genomes of 141 human-derived C. perfringens strains from public databases, this study conducted comprehensive analyses of antibiotic resistance genes, virulence genes, multilocus sequence typing (MLST), prophage detection, and pan-genome analysis for a total of 168 strains of C. perfringens. Antibiotics resistances among the isolates were phenotypically and genotypically analyzed and found 24 of them (88.9%, 24/27) were identified as multidrug-resistant (MDR). WGS analysis revealed that 13 strains belonged to known sequence types (ST), and the remaining strains represented 10 new STs. By analysis in combination with data of 141 C. perfringens isolates from the database, it was implied that ST221, ST72 and ST370 were present in both animal-derived and human-derived C. perfringens. It is worth noting that 108 out of 168 strains of C. perfringens (64.3%, 108/168) were found to carry prophages, which were found more prevalent in human-derived C. perfringens isolates. Pan-genome and phylogenetic analysis of 168 C. perfringens strains indicated that C. perfringens possesses an open pan-genome with genetic diversity. This study provides genomic insights into C. perfringens from food animals and humans, shedding light on the importance for monitoring the C. perfringens in livestock in China for better public health.

Keywords: Clostridium perfringens; multidrug-resistant bacteria; pangenome analysis; toxinotypes; whole genome sequencing.

FIGURE 1

Results of C. perfringens resistance analysis. (A) Heatmap: antimicrobial resistance phenotypes and predicted ARGs; Dendrogram: clustering rows with similar data. (B) Antibiotic resistance of each C. perfringens strain; (C) comparison of drug-resistant phenotypes and ARGs correlations in 27 strains of C. perfringens.

FIGURE 2

Results of virulence gene analysis. (A) Heatmap: prediction results of virulence genes; Dendrogram: clustering rows with similar data (red branches indicating strains of animal origin); (B) comparison of the number in virulence genes of C. perfringens from human and animal sources; (C) typing ratio of 168 C. perfringens strains.

FIGURE 3

Prophages in the C. perfringens genome by the prophages. (A) Heatmap: the prophage profile of 168 C. perfringens genomes; Dendrogram: clustering rows with similar data (red branches indicate strains of animal origin). (B) Genome sizes of complete, putative and incomplete prophages; (C) GC content of intact, questionable and incomplete prophages.

FIGURE 4

Phylogenetic tree based on the core genome of 24 strains of C. perfringens. The core genome of C. perfringens ATCC 13124 was used as outgroup. The six colors on the tree represent the six groups of analytical identification. The bootstrap values for each branch are all 1 (100%). The scale bar corresponds to 0.1 substitutions per site.

FIGURE 5

Results of pan-genomic analysis of C. perfringens. (A) Trend map of pan-genome; (B) the pan-genomes of these isolates were determined by comparing the pan-genomes, core genes, shell genes and cloud genes of the isolated C. perfringens; (C) phylogenetic tree analysis of the core genome of 168 strains of C. perfringens.