Clostridioides difficile infection (CDI) is the leading cause of life-threatening health care-related gastrointestinal illness worldwide. Phylogenetically appropriate closed reference genomes are essential for studies of C. difficile transmission and evolution. Here, we provide high-quality complete hybrid genome assemblies for the three most prevalent C. difficile strains causing CDI in Australia

Knight, Daniel R.

O\u27Grady, Keeley

Riley, Thomas V.

English

Research Online @ ECU

Edith Cowan University Research Online ECU Publications Post 2013 2021 Complete genome assemblies of three highly prevalent, toxigenic clostridioides difficile strains causing health care-associated infections in Australia Keeley O'Grady Thomas V. Riley Edith Cowan University Daniel R. Knight Follow this and additional works at: https://ro.ecu.edu.au/ecuworkspost2013 Digital Commons Network Logo  Part of the Diseases Commons 10.1128/MRA.00599-21 O’Grady, K., Riley, T. V., & Knight, D. R. (2021). Complete genome assemblies of three highly prevalent, toxigenic cl stridioides difficile strains causing health care-associated infections in Australia. Microbiology Resource Announcements, 10(31), article e00599-21.https://doi.org/10.1128/MRA.00599-21 This Other is posted at Research Online. https://ro.ecu.edu.au/ecuworkspost2013/11062 Complete Genome Assemblies of Three Highly Prevalent,Toxigenic Clostridioides difficile Strains Causing HealthCare-Associated Infections in AustraliaKeeley O’Grady,a Thomas V. Riley,a,b,c,d Daniel R. Knighta,baCentre for Biosecurity and One Health, Harry Butler Institute, Murdoch University, Murdoch, Western Australia, AustraliabSchool of Biomedical Sciences, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Western Australia, AustraliacSchool of Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia, AustraliadDepartment of Microbiology, PathWest Laboratory Medicine, Queen Elizabeth II Medical Centre, Nedlands, Western Australia, AustraliaABSTRACT Clostridioides difficile infection (CDI) is the leading cause of life-threateninghealth care-related gastrointestinal illness worldwide. Phylogenetically appropriate closedreference genomes are essential for studies of C. difficile transmission and evolution. Here,we provide high-quality complete hybrid genome assemblies for the three most prevalentC. difficile strains causing CDI in Australia.C lostridioides difficile causes life-threatening diarrhea and health care-related gastro-intestinal infections globally (1). Core genome single nucleotide polymorphism(cgSNP) analysis is highly discriminatory for bacterial transmission and outbreak detectionstudies and the gold standard for reconstructing large phylogenies of closely relatedmicrobes (2). A critical step in cgSNP analysis involves mapping raw sequence data to aclosely related reference genome, allowing for variant sites to be identified, filtered, andcompared between strains (3). Using phylogenetically related “closed” reference genomesprovides optimal mapping and variant calling. Australia has a diverse C. difficile populationdistinct from that of the rest of the world (1, 4), yet there are no phylogenetically appropri-ate reference genomes. Here, combining short- and long-read sequence technologies, weprovide high-quality complete genome sequences for three of the most prevalent C. difficilestrains causing C. difficile infection (CDI) in Australia, PCR ribotype 014 (RT014) (29.5% preva-lence), RT002 (11.8%), and RT056 (5.4%) (5, 6). Representative C. difficile strains of each ribotype(S-0352, S-0253, and S-0942, respectively) were selected from .1,500 isolates recovered frompatients with symptomatic CDI, part of the ongoing nationwide longitudinal surveillance ofCDI in Australia, the C. difficile Antimicrobial Resistance Surveillance (CDARS) study (5, 6).C. difficile strains from CDARS were cultured on blood agar in an anaerobic chamber(80% N2, 10% CO2, 10% H2) for 48 h (5). Total genomic DNA was extracted using aQuickGene DNA tissue kit (Kurabo Industries, Osaka, Japan) and used as input for bothshort-read (Illumina) and long-read (Oxford Nanopore Technologies [ONT]) sequencing.Illumina whole-genome sequencing (WGS) was performed using standard Nextera Flexpaired-end read (2 150-bp) libraries on an Illumina NovaSeq 6000 instrument (Illumina,San Diego, CA, USA) to an average read depth of 130. Default parameters were used forall software unless specified. The raw reads were filtered for quality (Q301) and adaptorsequences using Trim Galore v0.6.5 (https://github.com/FelixKrueger/TrimGalore). ONTsequencing was performed on a MinION Mk1C device (ONT, Oxford, UK) using an R9 gen-eration flow cell following a DNA by ligation protocol (SQK-LSK109). Filtlong v0.2.0 (https://github.com/rrwick/Filtlong) was used to filter the low-quality reads (keeping the top 90% ofreads and removing reads of ,1,000bp), resulting in 2.28 (S-0352), 2.59 (S-0253), and 6.66(S-0942) Gb of sequence data, respectively. The hybrid assembly of ONT and Illumina readswas performed using Unicycler v0.4.8 (7) with multiple rounds of polishing (Pilon v1.2.4,Citation O’Grady K, Riley TV, Knight DR. 2021.Complete genome assemblies of three highlyprevalent, toxigenic Clostridioides difficile strainscausing health care-associated infections inAustralia. Microbiol Resour Announc 10:e00599-21. https://doi.org/10.1128/MRA.00599-21.Editor David Rasko, University of MarylandSchool of MedicineCopyright © 2021 O’Grady et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to Daniel R. Knight,daniel.knight@murdoch.edu.au.Received 15 June 2021Accepted 9 July 2021Published 5 August 2021Volume 10 Issue 31 e00599-21 mra.asm.org 1GENOME SEQUENCESDownloaded from https://journals.asm.org/journal/mra on 03 October 2021 by 139.230.253.14.Racon v1.4.3) to improve the contiguity. Complete circular genomes were confirmed usingBandage v0.8.1 (8) and rotated to dnaA using Unicycler. The genomes were evaluated usingQUAST v2.344 (http://quast.sourceforge.net/quast) and annotated using the NCBI ProkaryoticGenome Annotation Pipeline v5.2 (9). The multilocus sequence type (ST) was determinedusing PubMLST (10).The summary genome features and metrics are shown in Table 1. A single 6,760-bpplasmid was identified in S-0253 (RT002). This data set increases the diversity of com-plete reference genomes available to the C. difficile research community, aiding futurestudies of C. difficile transmission and evolution.Data availability. The genome data are available at GenBank under BioProjectaccession number PRJNA734443 (complete genome assemblies) and at the ENA underBioProject accession number PRJEB41588 (Illumina sequence data); see Table 1 fordetails.ACKNOWLEDGMENTSK.O. was funded by an Australian Government Research Training Program Scholarship.D.R.K. was funded by a fellowship from the National Health and Medical Research Council(APP1138257).REFERENCES1. Knight DR, Elliott B, Chang BJ, Perkins TT, Riley TV. 2015. Diversity andevolution in the genome of Clostridium difficile. Clin Microbiol Rev28:721–741. https://doi.org/10.1128/CMR.00127-14.2. Eyre DW, Walker AS. 2013. Clostridium difficile surveillance: harnessingnew technologies to control transmission. Expert Rev Anti Infect Ther11:1193–1205. https://doi.org/10.1586/14787210.2013.845987.3. Olson ND, Lund SP, Colman RE, Foster JT, Sahl JW, Schupp JM, Keim P,Morrow JB, Salit ML, Zook JM. 2015. Best practices for evaluating singlenucleotide variant calling methods for microbial genomics. Front Genet6:235. https://doi.org/10.3389/fgene.2015.00235.4. Dingle KE, Elliott B, Robinson E, Griffiths D, Eyre DW, Stoesser N,Vaughan A, Golubchik T, Fawley WN, Wilcox MH, Peto TE, Walker AS,Riley TV, Crook DW, Didelot X. 2014. Evolutionary history of the Clostridiumdifficile pathogenicity locus. Genome Biol Evol 6:36–52. https://doi.org/10.1093/gbe/evt204.5. Knight DR, Giglio S, Huntington PG, Korman TM, Kotsanas D, Moore CV,Paterson DL, Prendergast L, Huber CA, Robson J, Waring L, Wehrhahn MC,Weldhagen GF, Wilson RM, Riley TV. 2015. Surveillance for antimicrobialresistance in Australian isolates of Clostridium difficile, 2013–14. J Antimi-crob Chemother 70:2992–2999. https://doi.org/10.1093/jac/dkv220.6. Hong S, Putsathit P, George N, Hemphill C, Huntington PG, Korman TM,Kotsanas D, Lahra M, McDougall R, Moore CV, Nimmo GR, Prendergast L,Robson J, Waring L, Wehrhahn MC, Weldhagen GF, Wilson RM, Riley TV,Knight DR. 2020. Laboratory-based surveillance of Clostridium difficileinfection in Australian healthcare and community settings, 2013 to 2018.J Clin Microbiol 58:e01552-20. https://doi.org/10.1128/JCM.01552-20.TABLE 1 Key features of C. difficile genomesFeatureData for strain:S-0352 S-0253 S-0942Strain epidemiologya RT014, ST2, clade 1 RT002, ST8, clade 1 RT056, ST34, clade 1Toxin profileb A1B1CDT2 A1B1CDT2 A1B1CDT2Originc Human, CDI, VIC 2014 Human, CDI, SA 2014 Human, CDI, SA 2016GenBank accession no. CP076377 CP076401, CP076402 CP076376ENA accession no. ERS5447138 ERS5447236 ERS5447376Genome size (bp) 4,251,987 4,089,134 (4,095,894d) 4,129,159%GC 28.96 28.52 28.71No. of CDSe 3,790 3,591 3,648No. of contigs 1 2 1No. of tRNAs 90 90 90No. of rRNAs 35 35 35No. of CRISPRsf 10 4 9Read metricsTotal no. of ONT reads 545,760 760,250 1,400,000Average ONT read length (bp, filtered) 5,518 4,663 9,038Total no. of Illumina reads (trimmed) 2,015,674 1,930,928 1,868,362a RT, PCR ribotype; ST, multilocus sequence type.b Presence/absence of full-length tcdA, tcdB (pathogenicity locus, PaLoc), and binary toxin cdtA/B (binary toxin locus, CdtLoc). CDT,C. difficile binary toxin.cVIC, Victoria; SA, South Australia,dCombined chromosome and plasmid length.eCDS, coding sequences.fCRISPRs, clustered regularly interspaced short palindromic repeats.O’Grady et al.Volume 10 Issue 31 e00599-21 mra.asm.org 2Downloaded from https://journals.asm.org/journal/mra on 03 October 2021 by 139.230.253.14.7. Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterialgenome assemblies from short and long sequencing reads. PLoS ComputBiol 13:e1005595. https://doi.org/10.1371/journal.pcbi.1005595.8. Wick RR, Schultz MB, Zobel J, Holt KE. 2015. Bandage: interactive visu-alization of de novo genome assemblies. Bioinformatics 31:3350–3352.https://doi.org/10.1093/bioinformatics/btv383.9. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L,Lomsadze A, Pruitt KD, BorodovskyM, Ostell J. 2016. NCBI Prokaryotic GenomeAnnotation Pipeline. Nucleic Acids Res 44:6614–6624. https://doi.org/10.1093/nar/gkw569.10. Griffiths D, Fawley W, Kachrimanidou M, Bowden R, Crook DW,Fung R, Golubchik T, Harding RM, Jeffery KJM, Jolley KA, Kirton R,Peto TE, Rees G, Stoesser N, Vaughan A, Walker AS, Young BC,Wilcox M, Dingle KE. 2010. Multilocus sequence typing of Clostrid-ium difficile. J Clin Microbiol 48:770–778. https://doi.org/10.1128/JCM.01796-09.Microbiology Resource AnnouncementVolume 10 Issue 31 e00599-21 mra.asm.org 3Downloaded from https://journals.asm.org/journal/mra on 03 October 2021 by 139.230.253.14.

Complete genome assemblies of three highly prevalent, toxigenic clostridioides difficile strains causing health care-associated infections in Australia

https://ro.ecu.edu.au/cgi/viewcontent.cgi?article=12068&context=ecuworkspost2013

Complete genome assemblies of three highly prevalent, toxigenic clostridioides difficile strains causing health care-associated infections in Australia

Abstract

Similar works

Full text

Available Versions

Research Online @ ECU