Genomic Analysis of Clostridium perfringens BEC/CPILE-Positive, Toxinotype D and E Strains Isolated from Healthy Children

Abstract

Clostridium perfringens toxinotype D, toxinotype E, and gastroenteritis-linked BEC/CPILE-positive strains have never been reported in healthy children. We isolated, whole-genome sequenced and bioinformatically characterised three C. perfringens isolates—type D (IQ1), type E (IQ2) and BEC/CPILE-positive (IQ3), recovered from the stools of three healthy two-year-olds, which were further compared to 128 C. perfringens genomes available from NCBI. The analysis uncovered a previously under-described putative toxin gene alv (alveolysin) encoded by isolates IQ2 and IQ3, which appeared to be a clade-specific trait associated with strains from domestic animals. A plasmid analysis indicated that the iota-toxin was encoded on a near-intact previously described plasmid pCPPB-1 in type E strain IQ2. The BEC genes becA and becB were carried on a near-identical pCPOS-1 plasmid previously associated with Japanese gastroenteritis outbreaks. Furthermore, a close phylogenetic relatedness was inferred between the French C. perfringens type E isolates cp515.17 and newly sequenced IQ2, suggesting geographical links. This study describes novel C. perfringens isolates from healthy individuals which encode important toxin genes, indicating the potential spread of these veterinary and clinically important strains and mobile genetic elements, and highlights areas for future research.

Comparative Genomics of Clostridium perfringens Reveals Patterns of Host-Associated Phylogenetic Clades and Virulence Factors

“…Alvoelysin ( alv ) was the only toxin limited to a single clade as all 35 strains that encoded alv were present in Clade V confirming a previous study showing it was clade-specific ( Kiu et al, 2019 ). Alveolysin is an understudied toxin of C. perfringens that is similar to perfringolysin ( Kiu et al, 2019 ) with both being cholesterol-dependent cytolysins, previously known as thiol-activated cytolysins ( Billington et al, 2000 ).…”

“…Alvoelysin (alv) was the only toxin limited to a single clade as all 35 strains that encoded alv were present in Clade V confirming a previous study showing it was cladespecific (Kiu et al, 2019). Alveolysin is an understudied toxin of C. perfringens that is similar to perfringolysin (Kiu et al, 2019) with both being cholesterol-dependent cytolysins, previously known as thiol-activated cytolysins (Billington et al, 2000). Gene duplications are frequent mutations in microbes (Reams and Roth, 2015), and we therefore hypothesize that alveolysin may have arisen from a gene duplication of perfringolysin followed by divergence during evolution as the two toxins are similar (~79% similarity) and generally encoded as little as 5 kb apart, although lateral gene transfer cannot be ruled out.…”

“…The only strain to encode binary enterotoxin ( becA and becB ), Q135.2 (IQ3), is also in clade V and was isolated from a fecal sample obtained from a healthy child ( Kiu et al, 2019 ). The becA and becB genes are plasmid-encoded and seem to be rare ( Kiu et al, 2019 ; Matsuda et al, 2019 ).…”

“…The only strain to encode binary enterotoxin (becA and becB), Q135.2 (IQ3), is also in clade V and was isolated from a fecal sample obtained from a healthy child (Kiu et al, 2019).…”

“…The becA and becB genes are plasmid-encoded and seem to be rare (Kiu et al, 2019;Matsuda et al, 2019).…”