Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens

Abstract

Background: Clostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome.

Results: Distinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis.

Conclusions: The high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. This study reveals that horizontal gene transfer appears to play a significant role in genetic variation of the C. perfringens chromosome as well as the plasmid content within strains.

Keywords: Adhesion; Capsule; Clostridium perfringens; Genome; Necrotic enteritis; Pangenome; Prophage.

Fig. 1

Presence/absence distribution of clusters. a. Histogram showing gene family distribution. b. Bar chart showing the number of new sequence clusters found in sequentially added C. perfringens genomes. c. Dot plot showing pan genome and core genome clusters in the C. perfringens genomes

Fig. 2

COG functional analysis of pan-genome. The histogram shows the predicted functionality of proteins assigned to core, accessory and singleton clusters

Fig. 3

PCA on the pan-matrix. Each dot marks a genome in the first two principal components of the pan-matrix. Strains are coloured by clade designation and disease-causing capability. Shapes represent; triangle, netB positive from diseased birds; squares, netB negative poultry isolates; circle, non-poultry netB negative isolate

Fig. 4

Total gene content phylogeny. Total gene content phylogeny. Tree of maximum likelihood based on total gene content of the chromosome of various C. perfringens strains. Red, Pink represent pathogenic strains of C. perfringens clades 1A, and 1B respectively, while orange represent pathogenic clade 2. Dark Green and light green represent non-pathogenic strains in clades 3 and 4 respectively. Black, positive; white, negative

Fig. 5

Presence of variable regions. Heatmap showing the presence and absence of variable chromosomal loci among 38 poultry strains and 21 non-poultry strains. Each column represents a different strain and each row represents a different chromosomal locus. Coloured sections represent a positive hit for that strain, grey represents a partial match (not entire locus but a single gene) and white is no match. Colours represent clade designation of a strains with pathogenic clade 1A (red), pathogenic clade 1B (pink) and pathogenic clade 2 (orange) while non-pathogenic clade 1 (dark green) and non-pathogenic clade 2 (light green). Non-poultry strains are coloured blue

Fig. 6

Adhesion locus variations. Genetic map showing sequence alignment of the different adhesion associated loci. Alignment is shown for four strains TAM-NE43, WER-NE36, EHE-NE18 and PBD1. TAM-NE43 has the intact VR-10A (blue) variant, WER-NE36 shows a highly deleted variation of VR-10A, VR-10B is represented by EHE-NE18 (green) [30] and PBD1 represents the VR-10C variant. Flanking genes of the locus sortase (srtB), and CPR0488 are coloured grey, and the two variants of the response regulator and sensor histidine kinase (SKH) are coloured red. Abbreviations: Alt exp.; alternative export protein, sipW; signal peptidase, hyp; hypothetical proteins, RR; response regulator, SHK; sensor histidine kinase, vWBFa; vonWillebrand factor A-like protein

Fig. 7

VR-10A variations. A genetic map showing sequence alignments of the different VR-10A adhesion loci. Alignment is shown for four strains: ATCC13124, SM101, TAM-NE43 and 13

Fig. 8

Capsule polysaccharide synthesis locus variations. Genetic map comparing the three capsule polysaccharide synthesis (CpCap) loci in pathogenic strains of C. perfringens. Representative regions from EHE-NE18 (mannose), TAM-NE46 (rhamnose) and EHE-NE7 (glucosamine) and the CpCPSL-B variants. a. Cds regions are colored as follows: Dark Blue – regulation, light blue – glycosyltransferase, red - UDP-mannose, orange – dTDP-rhamnose, Brown – UDP-L-fucose, Green – UDP-glucosamine/UDP-galactose, Dark pink – polysaccharide polymerase, yellow- polysaccharide transporter/flippase, Black – acetyltransferase, grey – hypothetical/unknown, Light Pink – mobile genetic elements and light green UDP-glycerol. b. As above but also Light Pink – phosphocholine

Fig. 9

Core genome phylogeny. Tree of maximum likelihood based on single nucleotide polymorphisms in the core chromosome of C. perfringens. Dark red and light red squares represent Pathogenic strains of C. perfringens clades 1 2 respectively while green triangles and diamonds represent non-pathogenic strains in clades 3 and 4 respectively. Black; positive, white; negative

Fig. 10

PFGE. Tree of PFGE fingerprints of various C. perfringens strains. Colours are as follows; white; negative, black; positive, for features including netB, VR-10A and isolation from chicken. Colours referring to clade designation are as follows; blue; Non-poultry, Green (dark and light); Non-pathogenic clade 3 and 4 respectively, Pink; pathogenic clade 1B, Red; pathogenic clade 1A, Orange; pathogenic clade 2