1887

Abstract

is a major enteric pathogen known to cause gastroenteritis in human adults. Although major outbreak cases are frequently reported, only limited whole-genome sequencing (WGS) based studies have been performed to understand the genomic epidemiology and virulence gene content of outbreak-associated strains. We performed phylogenomic analysis on 109 . isolates (human and food) obtained from disease cases in England and Wales between 2011 and 2017. Initial findings highlighted the enhanced discriminatory power of WGS in profiling outbreak strains, when compared to the current Public Health England referencing laboratory technique of fluorescent amplified fragment length polymorphism analysis. Further analysis identified that isogenic strains were associated with nine distinct care-home-associated outbreaks over the course of a 5-year interval, indicating a potential common source linked to these outbreaks or transmission over time and space. As expected, the enterotoxin gene was encoded in all but 4 isolates (96.3 %; 105/109), with virulence plasmids encoding (particularly pCPF5603 and pCPF4969 plasmids) extensively distributed (82.6 %; 90/109). Genes encoding accessory virulence factors, such as beta-2 toxin, were commonly detected (46.7 %; 51/109), and genes encoding phage proteins were also frequently identified. Overall, this large-scale genomic study of gastroenteritis-associated suggested that three major -encoding (toxinotype F) genotypes underlie these outbreaks: strains carrying (1) pCPF5603 plasmid, (2) pCPF4969 plasmid and (3) chromosomal- strains. Our findings substantially expanded our knowledge on type F involved in human-associated gastroenteritis, with further studies required to fully probe the dissemination and regional reservoirs of this enteric pathogen, which may help devise effective prevention strategies to reduce the food-poisoning disease burden in vulnerable patients, such as the elderly.

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2019-09-25
2019-10-21
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References

  1. Awad MM, Ellemor DM, Boyd RL, Emmins JJ, Rood JI. Synergistic effects of alpha-toxin and perfringolysin O in Clostridium perfringens-mediated gas gangrene. Infect Immun 2001;69:7904–7910 [CrossRef]
    [Google Scholar]
  2. Kiu R, Hall LJ. An update on the human and animal enteric pathogen Clostridium perfringens. Emerg Microbes Infect 2018;7:1–15 [CrossRef]
    [Google Scholar]
  3. Kiu R, Caim S, Alexander S, Pachori P, Hall LJ. Probing genomic aspects of the multi-host pathogen Clostridium perfringens reveals significant pangenome diversity, and a diverse array of virulence factors. Front Microbiol 2017;8:2485 [CrossRef]
    [Google Scholar]
  4. Revitt-Mills SA, Rood JI, Adams V. Clostridium perfringens extracellular toxins and enzymes: 20 and counting. Microbiol Aust 2015;114–117 [CrossRef]
    [Google Scholar]
  5. Kim YJ, Kim SH, Ahn J, Cho S, Kim D et al. Prevalence of Clostridium perfringens toxin in patients suspected of having antibiotic-associated diarrhea. Anaerobe 2017;48:34–36 [CrossRef]
    [Google Scholar]
  6. Sim K, Shaw AG, Randell P, Cox MJ, McClure ZE et al. Dysbiosis Anticipating necrotizing enterocolitis in very premature infants. Clin Infect Dis 2015;60:389–397 [CrossRef]
    [Google Scholar]
  7. Hobbs BC, Smith ME, Oakley CL, Warrack GH, Cruickshank JC. Clostridium welchii food poisoning. J Hyg 1953;51:75–101 [CrossRef]
    [Google Scholar]
  8. DuPont HL. Clinical practice. bacterial diarrhea. N Engl J Med 2009;361:1560–1569 [CrossRef]
    [Google Scholar]
  9. O'Brien SJ, Larose TL, Adak GK, Evans MR, Tam CC et al. Modelling study to estimate the health burden of foodborne diseases: cases, general practice consultations and hospitalisations in the UK, 2009. BMJ Open 2016;6:e011119 [CrossRef]
    [Google Scholar]
  10. Dolan GP, Foster K, Lawler J, Amar C, Swift C et al. An epidemiological review of gastrointestinal outbreaks associated with Clostridium perfringens, North East of England, 2012-2014. Epidemiol Infect 2016;144:1386–1393 [CrossRef]
    [Google Scholar]
  11. Adak GK, Long SM, O'Brien SJ. Trends in Indigenous foodborne disease and deaths, England and Wales: 1992 to 2000. Gut 2002;51:832–841 [CrossRef]
    [Google Scholar]
  12. Tam CC, Rodrigues LC, Viviani L, Dodds JP, Evans MR et al. Longitudinal study of infectious intestinal disease in the UK (IID2 study): incidence in the community and presenting to general practice. Gut 2012;61:69–77 [CrossRef]
    [Google Scholar]
  13. Borriello SP, Welch AR, Larson HE, Barclay F, Stringer MF et al. Enterotoxigenic Clostridium perfringens: a possible cause of antibiotic-associated diarrhoea. The Lancet 1984;323:305–307 [CrossRef]
    [Google Scholar]
  14. Larson HE, Borriello SP. Infectious diarrhea due to Clostridium perfringens. J Infect Dis 1988;157:390–391 [CrossRef]
    [Google Scholar]
  15. Food Standards Agency Foodborne Disease Strategy 2010-15 London: Food Standards Agency; 2011
    [Google Scholar]
  16. Food Standards Agency Report of the study of infectious intestinal disease in England. Commun Dis Rep CDR Wkly 2000;10:457
    [Google Scholar]
  17. Rood JI, Adams V, Lacey J, Lyras D, McClane BA et al. Expansion of the Clostridium perfringens toxin-based typing scheme. Anaerobe 2018;53:5–10 [CrossRef]
    [Google Scholar]
  18. Fernández Miyakawa ME, Pistone Creydt V, Uzal FA, McClane BA, Ibarra C. Clostridium perfringens enterotoxin damages the human intestine in vitro. Infect Immun 2005;73:8407–8410 [CrossRef]
    [Google Scholar]
  19. Shinoda T, Shinya N, Ito K, Ohsawa N, Terada T et al. Structural basis for disruption of claudin assembly in tight junctions by an enterotoxin. Sci Rep 2016;6:33632 [CrossRef]
    [Google Scholar]
  20. Ronco T, Stegger M, Ng KL, Lilje B, Lyhs U et al. Genome analysis of Clostridium perfringens isolates from healthy and necrotic enteritis infected chickens and turkeys. BMC Res Notes 2017;10:270 [CrossRef]
    [Google Scholar]
  21. Gaucher M-L, Perron GG, Arsenault J, Letellier A, Boulianne M et al. Recurring necrotic enteritis outbreaks in commercial broiler chicken flocks strongly influence toxin gene carriage and species richness in the resident Clostridium perfringens population. Front Microbiol 2017;8:881 [CrossRef]
    [Google Scholar]
  22. Verherstraeten S, Goossens E, Valgaeren B, Pardon B, Timbermont L et al. The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells. Vet Res 2013;44:45 [CrossRef]
    [Google Scholar]
  23. Lacey JA, Allnutt TR, Vezina B, Van TTH, Stent T et al. Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens. BMC Genomics 2018;19:379 [CrossRef]
    [Google Scholar]
  24. Mahamat Abdelrahim A, Radomski N, Delannoy S, Djellal S, Le Négrate M et al. Large-Scale Genomic Analyses and Toxinotyping of Clostridium perfringens Implicated in Foodborne Outbreaks in France. Front Microbiol 2019;10:777 [CrossRef]
    [Google Scholar]
  25. Amar CFL, East CL, Grant KA, Gray J, Iturriza-Gomara M et al. Detection of viral, bacterial, and parasitological RNA or DNA of nine intestinal pathogens in fecal samples archived as part of the English infectious intestinal disease study: assessment of the stability of target nucleic acid. Diagn Mol Pathol 2005;14:90–96 [CrossRef]
    [Google Scholar]
  26. Roussel S, Félix B, Grant K, Dao TT, Brisabois A et al. Fluorescence amplified fragment length polymorphism compared to pulsed field gel electrophoresis for Listeria monocytogenes subtyping. BMC Microbiol 2013;13:14 [CrossRef]
    [Google Scholar]
  27. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012;19:455–477 [CrossRef]
    [Google Scholar]
  28. Page AJ, De Silva N, Hunt M, Quail MA, Parkhill J et al. Robust high-throughput prokaryote de novo assembly and improvement pipeline for Illumina data. Microb Genom 2016;2:e000083 [CrossRef]
    [Google Scholar]
  29. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014;30:2068–2069 [CrossRef]
    [Google Scholar]
  30. Davis MPA, van Dongen S, Abreu-Goodger C, Bartonicek N, Enright AJ. Kraken: a set of tools for quality control and analysis of high-throughput sequence data. Methods 2013;63:41–49 [CrossRef]
    [Google Scholar]
  31. Pritchard L, Glover RH, Humphris S, Elphinstone JG, Toth IK. Genomics and taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens. Anal Methods 2016;8:12–24 [CrossRef]
    [Google Scholar]
  32. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 2017;27:722–736 [CrossRef]
    [Google Scholar]
  33. Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics 2015;31:3691–3693 [CrossRef]
    [Google Scholar]
  34. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 2013;30:772–780 [CrossRef]
    [Google Scholar]
  35. Page AJ, Taylor B, Delaney AJ, Soares J, Seemann T et al. SNP-sites: rapid efficient extraction of SNPs from multi-FASTA alignments. Microb Genom 2016;2:e000056 [CrossRef]
    [Google Scholar]
  36. Price MN, Dehal PS, Arkin AP. FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One 2010;5:e9490 [CrossRef]
    [Google Scholar]
  37. Letunic I, Bork P. Interactive tree of life (iTOL) V3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res 2016;44:W242–W245 [CrossRef]
    [Google Scholar]
  38. Seemann T, Klotzl F, Page AJ. snp-dists. 0.2 ed2018. Convert a FASTA alignment to SNP distance matrix 2018
  39. Tonkin-Hill G, Lees JA, Bentley SD, Frost SDW, Corander J. RhierBAPS: an R implementation of the population clustering algorithm hierBAPS. Wellcome Open Res 2018;3:93 [CrossRef]
    [Google Scholar]
  40. Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P et al. Card 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res 2017;45:D566–D573 [CrossRef]
    [Google Scholar]
  41. Seemann T. ABRicate. 0.5 ed2018. Mass screening of contigs for antimicrobial resistance or virulence genes 2018
  42. Hunt M, Mather AE, Sánchez-Busó L, Page AJ, Parkhill J et al. ARIBA: rapid antimicrobial resistance genotyping directly from sequencing reads. Microb Genom 2017;3:e000131 [CrossRef]
    [Google Scholar]
  43. Roosaare M, Puustusmaa M, Möls M, Vaher M, Remm M. PlasmidSeeker: identification of known plasmids from bacterial whole genome sequencing reads. PeerJ 2018;6:e4588 [CrossRef]
    [Google Scholar]
  44. Brynildsrud O, Bohlin J, Scheffer L, Eldholm V. Rapid scoring of genes in microbial pan-genome-wide association studies with Scoary. Genome Biol 2016;17:238 [CrossRef]
    [Google Scholar]
  45. Huerta-Cepas J, Forslund K, Coelho LP, Szklarczyk D, Jensen LJ et al. Fast genome-wide functional annotation through orthology assignment by eggNOG-mapper. Mol Biol Evol 2017;34:2115–2122 [CrossRef]
    [Google Scholar]
  46. Arndt D, Marcu A, Liang Y, Wishart DS. PHAST, PHASTER and PHASTEST: tools for finding prophage in bacterial genomes. Brief Bioinform 2017;4:bbx121 [CrossRef]
    [Google Scholar]
  47. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P et al. Artemis: sequence visualization and annotation. Bioinformatics 2000;16:944–945 [CrossRef]
    [Google Scholar]
  48. Sullivan MJ, Petty NK, Beatson SA. Easyfig: a genome comparison visualizer. Bioinformatics 2011;27:1009–1010 [CrossRef]
    [Google Scholar]
  49. Lahti P, Heikinheimo A, Johansson T, Korkeala H. Clostridium perfringens type A strains carrying a plasmid-borne enterotoxin gene (genotype IS1151-cpe or IS1470-like-cpe) as a common cause of food poisoning. J Clin Microbiol 2008;46:371–373 [CrossRef]
    [Google Scholar]
  50. Theoret JR, Li J, Navarro MA, Garcia JP, Uzal FA et al. Native or proteolytically activated NanI sialidase enhances the binding and cytotoxic activity of Clostridium perfringens enterotoxin and beta toxin. Infect Immun 2018;86:e00730-17 [CrossRef]
    [Google Scholar]
  51. Larcombe S, Hutton ML, Lyras D. Involvement of bacteria other than Clostridium difficile in antibiotic-associated diarrhoea. Trends Microbiol 2016;24:463–476 [CrossRef]
    [Google Scholar]
  52. Fisher DJ, Miyamoto K, Harrison B, Akimoto S, Sarker MR et al. Association of beta2 toxin production with Clostridium perfringens type A human gastrointestinal disease isolates carrying a plasmid enterotoxin gene. Mol Microbiol 2005;56:747–762 [CrossRef]
    [Google Scholar]
  53. Sparks SG, Carman RJ, Sarker MR, McClane BA. Genotyping of enterotoxigenic Clostridium perfringens fecal isolates associated with antibiotic-associated diarrhea and food poisoning in North America. J Clin Microbiol 2001;39:883–888 [CrossRef]
    [Google Scholar]
  54. Tanaka D, Isobe J, Hosorogi S, Kimata K, Shimizu M et al. An outbreak of food-borne gastroenteritis caused by Clostridium perfringens carrying the cpe gene on a plasmid. Jpn J Infect Dis 2003;56:137–139
    [Google Scholar]
  55. Li J, McClane BA. Further comparison of temperature effects on growth and survival of Clostridium perfringens type A isolates carrying a chromosomal or plasmid-borne enterotoxin gene. Appl Environ Microbiol 2006;72:4561–4568 [CrossRef]
    [Google Scholar]
  56. Collie RE, McClane BA. Evidence that the enterotoxin gene can be episomal in Clostridium perfringens isolates associated with non-food-borne human gastrointestinal diseases. J Clin Microbiol 1998;36:30–36
    [Google Scholar]
  57. Lindström M, Heikinheimo A, Lahti P, Korkeala H. Novel insights into the epidemiology of Clostridium perfringens type A food poisoning. Food Microbiol 2011;28:192–198 [CrossRef]
    [Google Scholar]
  58. Tanaka D, Kimata K, Shimizu M, Isobe J, Watahiki M et al. Genotyping of Clostridium perfringens isolates collected from food poisoning outbreaks and healthy individuals in Japan based on the cpe locus. Jpn J Infect Dis 2007;60:68–69
    [Google Scholar]
  59. Cornillot E, Saint-Joanis B, Daube G, Katayama S, Granum PE et al. The enterotoxin gene (cpe) of Clostridium perfringens can be chromosomal or plasmid-borne. Mol Microbiol 1995;15:639–647 [CrossRef]
    [Google Scholar]
  60. Miyamoto K, Fisher DJ, Li J, Sayeed S, Akimoto S et al. Complete sequencing and diversity analysis of the enterotoxin-encoding plasmids in Clostridium perfringens type A non-food-borne human gastrointestinal disease isolates. J Bacteriol 2006;188:1585–1598 [CrossRef]
    [Google Scholar]
  61. Brynestad S, Sarker MR, McClane BA, Granum PE, Rood JI. Enterotoxin plasmid from Clostridium perfringens is conjugative. Infect Immun 2001;69:3483–3487 [CrossRef]
    [Google Scholar]
  62. Gera K, Le T, Jamin R, Eichenbaum Z, McIver KS. The phosphoenolpyruvate phosphotransferase system in group A Streptococcus acts to reduce streptolysin S activity and lesion severity during soft tissue infection. Infect Immun 2014;82:1192–1204 [CrossRef]
    [Google Scholar]
  63. Genevaux P, Wawrzynow A, Zylicz M, Georgopoulos C, Kelley WL. DjlA is a third DnaK co-chaperone of Escherichia coli, and DjlA-mediated induction of colanic acid capsule requires DjlA-DnaK interaction. J Biol Chem 2001;276:7906–7912 [CrossRef]
    [Google Scholar]
  64. Barocchi MA, Ries J, Zogaj X, Hemsley C, Albiger B et al. A pneumococcal pilus influences virulence and host inflammatory responses. Proc Natl Acad Sci U S A 2006;103:2857–2862 [CrossRef]
    [Google Scholar]
  65. Arita-Morioka K-ichi, Yamanaka K, Mizunoe Y, Ogura T, Sugimoto S. Novel strategy for biofilm inhibition by using small molecules targeting molecular chaperone DnaK. Antimicrob Agents Chemother 2015;59:633–641 [CrossRef]
    [Google Scholar]
  66. Boyd EF. Bacteriophage-encoded bacterial virulence factors and phage-pathogenicity island interactions. Adv Virus Res 2012;82:91–118 [CrossRef]
    [Google Scholar]
  67. Kinney DM, Bramucci MG. Analysis of Bacillus subtilis sporulation with spore-converting bacteriophage PMB12. J Bacteriol 1981;145:1281–1285
    [Google Scholar]
  68. Fortier L-C, Sekulovic O. Importance of prophages to evolution and virulence of bacterial pathogens. Virulence 2013;4:354–365 [CrossRef]
    [Google Scholar]
  69. Li J, McClane BA. Contributions of NanI sialidase to Caco-2 cell adherence by Clostridium perfringens type A and C strains causing human intestinal disease. Infect Immun 2014;82:4620–4630 [CrossRef]
    [Google Scholar]
  70. Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A et al. Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci U S A 2002;99:996–1001 [CrossRef]
    [Google Scholar]
  71. Andersen JL, He G-X, Kakarla P, K C R, Kumar S et al. Multidrug efflux pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus bacterial food pathogens. Int J Environ Res Public Health 2015;12:1487–1547 [CrossRef]
    [Google Scholar]
  72. Govind R, Dupuy B. Secretion of Clostridium difficile toxins A and B requires the holin-like protein TcdE. PLoS Pathog 2012;8:e1002727 [CrossRef]
    [Google Scholar]
  73. Lakshminarayanan B, Harris HMB, Coakley M, O'Sullivan O, Stanton C et al. Prevalence and characterization of Clostridium perfringens from the faecal microbiota of elderly Irish subjects. J Med Microbiol 2013;62:457–466 [CrossRef]
    [Google Scholar]
  74. Hu W-S, Kim H, Koo OK. Molecular genotyping, biofilm formation and antibiotic resistance of enterotoxigenic Clostridium perfringens isolated from meat supplied to school cafeterias in South Korea. Anaerobe 2018;52:115–121 [CrossRef]
    [Google Scholar]
  75. Li J, McClane BA. A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates. PLoS Pathog 2008;4:e1000056 [CrossRef]
    [Google Scholar]
  76. Ashton PM, Nair S, Peters TM, Bale JA, Powell DG et al. Identification of Salmonella for public health surveillance using whole genome sequencing. PeerJ 2016;4:e1752 [CrossRef]
    [Google Scholar]
  77. Deguchi A, Miyamoto K, Kuwahara T, Miki Y, Kaneko I et al. Genetic characterization of type A enterotoxigenic Clostridium perfringens strains. PLoS One 2009;4:e5598 [CrossRef]
    [Google Scholar]
  78. Claesson MJ, Jeffery IB, Conde S, Power SE, O'Connor EM et al. Gut microbiota composition correlates with diet and health in the elderly. Nature 2012;488:178–184 [CrossRef]
    [Google Scholar]
  79. Schmid D, Allerberger F, Huhulescu S, Pietzka A, Amar C et al. Whole genome sequencing as a tool to investigate a cluster of seven cases of listeriosis in Austria and Germany, 2011-2013. Clin Microbiol Infect 2014;20:431–436 [CrossRef]
    [Google Scholar]
  80. Keto-Timonen R, Heikinheimo A, Eerola E, Korkeala H. Identification of Clostridium species and DNA fingerprinting of Clostridium perfringens by amplified fragment length polymorphism analysis. J Clin Microbiol 2006;44:4057–4065 [CrossRef]
    [Google Scholar]
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