| Clostridium perfringens(C.perfringens)is an important zoonotic pathogen that exists in soil,sewage,and the intestines of humans and animals,and is the most widely distributed potential pathogenic microorganism in nature One of them,can cause a variety of diseases in humans and animals,including enterotoxemia(Enterotoxemia),hemorrhagic and necrotizing enteritis(NE),traumatic wound infection,cellulitis(Cellulitis)and myonecrosis,Post-abortion sepsis,intravascular hemolysis and postpartum infection(Puerperal fever),etc.,seriously endanger human and animal health.At present,the research on C.perfringens by conventional methods has been relatively detailed,but there are few related studies on the analysis of C.perfringens from the whole genome level.The study of the genomic diversity of C.perfringens isolates contributes to our understanding of the differences and relatedness of pathogenic and non-pathogenic strains,the identification of host-and disease-specific determinants,and the establishment of disease prevention Strategies for future platforms such as vaccines,bacteriophage therapy,antibiotic therapy,etc.In this study,an epidemiological investigation was conducted on the outbreak of the disease with clinical symptoms such as diarrhea and abdominal distension in elk(Pere David’s deer)in Shishou Elk Reserve,Hubei Province.Whole-genome sequencing was performed and comparative genome analysis was performed with whole-genome data of 8 C.perfringens selected from the NCBI Genome database(https://www.ncbi.nlm.nih.gov/genome/),from molecular The gene composition and genetic evolution relationship of the virulent strain CX1-4 were studied at the level.The result is as follows:1.Isolation,identification and screening of Clostridium perfringensThe diseased elk had clinical symptoms such as soy sauce-colored feces and rapid abdominal swelling after death.The autopsy showed that tissues and organs were enlarged,with varying degrees of congestion and bleeding on the surface.Bulbar telangiectasia and congestion,splenic lymphocyte necrosis.A total of 5 strains of Clostridium perfringens were isolated from the tissues and organs of the dead elk and named as CX1-1,CX1-3,CX1-4,CX2-1 and CX2-2.After multiple PCR identification,the results showed that All were type A;the biochemical identification results were consistent with the biochemical characteristics of Clostridium perfringens in the Berger Bacterial Identification Manual 8th Edition.The growth curve,the fermentation test of milk medium,and the puncture test of trisaccharide iron showed that the isolate grew faster in 0-5h,and reached the plateau stage after 5h.During the growth process,acid production and gas production can all make the milk medium burst and ferment.The results of drug susceptibility test showed that the isolates were sensitive to β-lactams,tetracyclines and other antibiotics,and were resistant to sulfonamide antibiotics;the results of phospholipase C(PLC)activity assay showed that strain CX1-4 PLC had the strongest activity,which was the same as the control group.significant difference.The C.perfringens CX1-4 strain was screened as a candidate strain for whole genome sequencing by measuring the hemolytic activity of the isolated strain and the mouse median lethal dose(LD50)test combined with the results of the PLC activity test.The most virulent strain C.perfringens CX1-4 was used to challenge mice,and the mice in the experimental group developed clinical symptoms such as depression and disheveled coat.2.CX1-4 whole genome sequencing and analysisIllumina NovaSeq and PromethION sequencer sequencing platforms were used to sequence the virulent strain C.perfringens CX1-4 in the second and third generation.The sequencing results were filtered,assembled and annotated.The GC ratio was 28.36%,the full length of the plasmid sequence was 47148bp,and the GC ratio was 27.33%.The chromosome sequence contained 3193 genes,3002 CDS regions,94 tRNA coding sequences,and 30 rRNA coding sequences(10 5S,10 16S,10 23S),genome analysis also found 72 pseudogenes and 4 gene islands,but no CPISPR sequence was found.In the general database annotation,the Gene Ontology classification results showed that the C.perfringens 1-4 genome had the largest number of genes related to biological processes,with 1494 genes,mainly involved in protein translation,spore and cell wall formation;KEGG Pathway classification results showed that the genome Nearly half(1557,48.76%)of the genes were related to the KEGG metabolic pathway,involved in cellular processes,environmental information processing,genetic information processing,metabolism,and body systems(organismal systems),most of which(1108)genes are involved in the metabolic processes of carbohydrates,nucleotides,enzymes and vitamins,and the least involved in cellular processes,only 1.54%.There are 1300 gene annotation results in the COG classification results,the number of genes involved in translation,ribosome structure and biosynthesis is the largest(147),and the cytoskeleton-related genes are the least,accounting for only 0.15%.From the VFDB annotation results,it can be seen that C.perfringens carries 18 virulence genes in addition to the cpe and nagK genes among the genes related to the virulence factors of C.perfringens,which further explains the reason why the CX1-4 strain showed strong virulence in the test.3.Comparative genomic analysis of virulent strain CX1-4The genomes of the screened virulent strain CX1-4 were compared with the whole genomes of 8 reference strains selected from the NCBI Genome database.The results showed that the genome size of each strain was between 2.9 and 4.0M in terms of the basic characteristics of the genome.The ratio is about 28%,and the number of rRNA genes in C.perfringens ATCC 13124 strain is 24,which is different from other strains.The results of 16S rRNA phylogenetic tree showed that C.perfringens CX1-4 was closest to C.perfringens SM101 in terms of genetic evolution,but had a lower branch bootstrap value and was farther away from C.perfringens EHE-NE18.In the analysis results of genome ANI value,the ANI value between C.perfringens CX1-4 and the whole genome of the Australian chicken-derived strain EHE-NE18 was the lowest,only 95.33%,and the average ANI value among each type A strain was 97.30%.The results of genome collinearity showed that,except C.perfringens EHE-NE18,the genomes of other strains had good homology and gene consistency.In the comparison results of virulence factors,only C.perfringens CX1-4 contains cna virulence genes,and has the largest number of virulence factor-related genes(18),and the four C.perfringens have a total of 7 virulence genes(virS,virR,plc,colA,fbpA/fbp68,cloS1,Hemolysin),CX1-4 and SM101 each have a specific virulence gene,respectively cna and cpe.The above experiments showed that in this study,5 strains of Clostridium perfringens type A were isolated from the tissue samples of elk.Genome sequencing analysis,through comparative genome analysis of the genetic variation of its genome,to screen out the specific virulence gene cna.This research is expected to lay the foundation for the study of the molecular mechanism of C.perfringens pathogenicity and the establishment of future platforms for disease prevention strategies,such as vaccines,phage therapy,and antibiotic treatment. |
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