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Phylogenetic and genomic analysis reveals high genomic openness and genetic diversity of Clostridium perfringens - PubMed

Phylogenetic and genomic analysis reveals high genomic openness and genetic diversity of Clostridium perfringens

Yuqing Feng et al. Microb Genom. 2020 Oct.

Abstract

Clostridium perfringens is associated with a variety of diseases in both humans and animals. Recent advances in genomic sequencing make it timely to re-visit this important pathogen. Although the genome sequence of C. perfringens was first determined in 2002, large-scale comparative genomics with isolates of different origins is still lacking. In this study, we used whole-genome sequencing of 45 C. perfringens isolates with isolation time spanning an 80-year period and performed comparative analysis of 173 genomes from worldwide strains. We also conducted phylogenetic lineage analysis and introduced an openness index (OI) to evaluate the openness of bacterial genomes. We classified all these genomes into five lineages and hypothesized that the origin of C. perfringens dates back to ~80 000 years ago. We showed that the pangenome of the 173 C. perfringens strains contained a total of 26 954 genes, while the core genome comprised 1020 genes, accounting for about a third of the genome of each isolate. We demonstrated that C. perfringens had the highest OI compared with 51 other bacterial species. Intact prophage sequences were found in nearly 70.0 % of C. perfringens genomes, while CRISPR sequences were found only in ~40.0 %. Plasmids were prevalent in C. perfringens isolates, and half of the virulence genes and antibiotic resistance genes (ARGs) identified in all the isolates could be found in plasmids. ARG-sharing network analysis showed that C. perfringens shared its 11 ARGs with 55 different bacterial species, and a high frequency of ARG transfer may have occurred between C. perfringens and species in the genera Streptococcus and Staphylococcus. Correlation analysis showed that the ARG number in C. perfringens strains increased with time, while the virulence gene number was relative stable. Our results, taken together with previous studies, revealed the high genome openness and genetic diversity of C. perfringens and provide a comprehensive view of the phylogeny, genomic features, virulence gene and ARG profiles of worldwide strains.

Keywords: Antibiotic Resistance Gene; Clostridium perfringens; Openness Index; Phylogenetic Tree; Toxins.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1.
Fig. 1.

Phylogenetic assignment of

C. perfringens isolates and validation. (a) Bayesian-dated phylogeny of C. perfringens . We classified the 173 C . perfringens genomes into five lineages according to their phylogenetic relationships. The five lineages are labelled with different colours. Information about location, host and diseases are shown to the right of the phylogenetic tree. (b) PCA of core genome sequences in 173 strains. (c) Pairwise F ST values between the four lineages. (d) Pairwise SNP distances within and between isolates of the four lineages.
Fig. 2.
Fig. 2.

Openness of the

C. perfringens genome. (a) Proportions of accessary genes and core genes in the pangenome. (b) Accumulation of the pan genes and core genes with increasing genome number. (c) Comparison of the C. perfringens genome OI with 51 other bacterial species. (d) Spearman's correlation between bacterial genome OI and GC contents of each species. (e) Spearman's correlation between median values of the bacterial genome OI and median values of 16S rRNA gene copy number.
Fig. 3.
Fig. 3.

Prophages in the

C. perfringens genome and the toxins carried by the prophages. (a) Heatmap visualizing the prophage profile of 173 C . perfringens genomes. Cells in purple and white indicate the presence and absence of prophages, respectively. (b) Toxin genes carried by prophages in 16 isolates.
Fig. 4.
Fig. 4.

Virulence genes in the

C. perfringens genome. (a) Heatmap visualizing the virulence gene profile of 173 C . perfringens genomes. Cells in purple and white indicate the presence and absence of genes, respectively. (b) The virulence gene number in each lineage. (c) The identity of the nucleotide sequences of the seven toxinotyping genes. (d) K a/K s values of all the seven toxins.
Fig. 5.
Fig. 5.

ARGs in the

C. perfringens genome and their transfer network among different bacterial genomes. (a) Heatmap visualizing the ARG profile of 173 C . perfringens genomes. Cells in purple and white indicate the presence and absence of genes, respectively. (b) ARG number in each lineage. (c) ARG number in strains from different continents (AF: Africa; AS: Asia; EU: Europe; NA: North America; OA: Oceania). (d) The ARG transfer network between C. perfringens and other species. The cyan octagon represents C. perfringens ; nodes with a brown circle represent ARGs found in the genomes of C. perfringens ; nodes with squares represent bacterial species sharing (>99.0 % nucleotide identity) ARGs with C. perfringens (purple: species from Firmicutes ; pink: species from Proteobacteria ; green: species from Actinobacteria ).
Fig. 6.
Fig. 6.

Prediction of the locations of virulence and antibiotic resistance genes. Purple and pink cells indicate chromosome and plasmid locations, respectively.

Fig. 7.
Fig. 7.

The changes of ARGs and virulence factors (VFs) in

C. perfringens over time. The correlation of ARG (a) and VF (b) number with strain isolation times. (c) The correlation between ARG number and VF number in different strains. Dot size is proportional to the number of strains. (d) Changes in the total number of ARGs and VFs in C. perfringens over time.

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