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Hall, Lindsay J.

Kiu, Raymond

Frontiers in Microbiology

Raymond Kiu

Lindsay J. Hall

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Response: Commentary: Probing Genomic Aspects of the Multi-Host Pathogen Clostridium perfringens Reveals Significant Pangenome Diversity, and a Diverse Array of Virulence Factors

Strous, Heather, (Executive Editor)

Williams, Sue, (Faculty Advisor)

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GlimmerGlass Volume 59 Number 02 (1999)

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GlimmerGlass Volume 58 Number 07 (1999)

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GlimmerGlass Volume 59 Number 08 (2000)

GlimmerGlass Volume 59 Number 03-04 (1999)

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GlimmerGlass Volume 59 Number 07 (2000)

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GENERAL COMMENTARYpublished: 16 August 2018doi: 10.3389/fmicb.2018.01857Frontiers in Microbiology | www.frontiersin.org 1 August 2018 | Volume 9 | Article 1857Edited by:John R. Battista,Louisiana State University,United StatesReviewed by:Karl Hassan,University of Newcastle, Australia*Correspondence:Lindsay J. HallLindsay.Hall@quadram.ac.ukSpecialty section:This article was submitted toEvolutionary and GenomicMicrobiology,a section of the journalFrontiers in MicrobiologyReceived: 02 June 2018Accepted: 24 July 2018Published: 16 August 2018Citation:Kiu R and Hall LJ (2018) Response:Commentary: Probing GenomicAspects of the Multi-Host PathogenClostridium perfringens RevealsSignificant Pangenome Diversity, anda Diverse Array of Virulence Factors.Front. Microbiol. 9:1857.doi: 10.3389/fmicb.2018.01857Response: Commentary: ProbingGenomic Aspects of the Multi-HostPathogen Clostridium perfringensReveals Significant PangenomeDiversity, and a Diverse Array ofVirulence FactorsRaymond Kiu 1,2 and Lindsay J. Hall 1*1Gut Microbes and Health Programme, Quadram Institute Bioscience, Norwich Research Park, Norwich, United Kingdom,2Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, United KingdomKeywords: pangenome, antimicrobial resistance, Clostridium perfringens, genomics, whole genome sequencing,Clostridial infection, exotoxins, pathogenA Commentary onCommentary: Probing Genomic Aspects of the Multi-Host Pathogen Clostridium perfringensReveals Significant Pangenome Diversity, and a Diverse Array of Virulence Factorsby Gohari, I. M., and Prescott, J. F. (2018). Front. Microbiol. 9:1856. doi: 10.3389/fmicb.2018.01856We firstly would like to express our appreciation for the comments and efforts made by Gohariand Prescott regarding their detailed commentary of our Clostridium perfringens Whole GenomeSequencing (WGS) based genomic and phylogenetic study we published in December 2017. Weare particularly pleased to note that the authors acknowledge that our “work has contributedsignificantly to understanding of genomic diversity of this bacterium.”The aim of our study was to conduct a robust large-scaleWGS study on this important pathogen,and to highlight genomic insights to the global scientific community. We believe that WGS couldprove extremely useful in exploring new traits in C. perfringens, as has been performed in manydifferent bacterial pathogens. We agree with the authors, and indeed highlight this throughout ouroriginal study, that alongside genomic-based studies, clinical metadata, epidemiological studies,and phenotypic testing, will be central to determine the impact of genetic variation in C. perfringensin the context of human and animal health.We would first like to respond to the authors commentary suggesting a “clear mistake” or“incorrect conclusion” in that netB gene (a gene that encode NetB toxin, which is associatedwith avian Necrotising Enteritis) has no role in canine haemorrhagic gastroenteritis andfoal necrotising enteritis (Keyburn et al., 2008, 2010; Rood et al., 2016). We appreciate theauthors have a track record of working in this particular area of C. perfringens virulence, ashighlighted by citation of their own work throughout the commentary. They highlight thatnetB and netE genes (which encodes proteins NetB and NetE respectively) share high sequenceidentity of 78% amino acid (according to their published work in 2015, 79% amino acidsequence identity; Mehdizadeh Gohari et al., 2015), which may have potentially contributedto our “understandable misinterpretation” of the data. Based on our informatics filteringparameters, we also determined that netB and netE are highly identical and not distinctiveKiu and Hall Genomic Analysis of Clostridium perfringensFIGURE 1 | (A) A BLASTn sequence alignment performed on a local machine that aligns epsilon toxin gene etx (NCBI accession: M95206.1) with PacBio-sequencedNCTC8503 previously assembled genome (ENA accession: SAMEA3879480; contig 5: nucleotide position 51607-52593). Only a point mutation at position 762 isdetected with 100% coverage and > 99% sequence identity. (B) Toxin profile of NCTC8503 isolate, performed via sequence similarity search (“best-match” approach)pipeline ABRicate (https://github.com/tseemann/abricate; BLASTn-based tool) that confirmed the presence of etx gene in NCTC8503 genome with 99.90% identity(at 100% coverage).at nucleotide sequence level. This was based on strict double-filtering strategy at 80% identity and E-value of 10−20, (routinelyapplied in WGS studies to infer identical genes; Pearson, 2013;Kiu et al., 2017), and was used as we were undertaking aglobal in silico-based approach to explore a significant numberof C. perfringens genomes for virulence-associated traits (notsolely focused on these two toxin genes). Consequently, wehave reanalysed the data and determined that a higher sequenceFrontiers in Microbiology | www.frontiersin.org 2 August 2018 | Volume 9 | Article 1857Kiu and Hall Genomic Analysis of Clostridium perfringensidentity threshold at 90% (BLASTn) confirms an absence ofthe netB gene in these NetF-associated genomes. We thankthe authors for highlighting the high sequence similarly ofthese toxin genes, and will factor these parameters in forfuture studies (Camacho et al., 2009). As we carried outa purely bioinformatic-based study, and are aware of thesensitivity/specificity of different computational pipelines andparameters, we were careful not to come to any definitive“conclusions,” and “suggested” that NetB toxin “might beinvolved,” thus clarifying our discussion points. Notably, withthe advancement in bioinformatics tools, differentiation betweenhighly similar genes may be possible using a “best-match”approach to avoid inaccurate annotations.To address the question as to whether or not NCTC8503 isan “NE isolate,” we have re-traced the source of this isolate andits sequence data. NCTC8503 is a C. perfringens strain isolatedby Bennetts (1930) according to the history record shown onthe Public Health England NCTC official website1 Nairn andBamford (1967) state “Bennetts (1930) had earlier reported theisolation of Bacillus welchii from a bowel lesion in a BlackOrpington pullet with intestinal coccidiosis, and he consideredthat an enterotoxaemia had contributed to the bird’s death”;B. welchii was later renamed as C. perfringens. Furthermore,Williams et al. states “A frequent, although sporadic, poultryclostridiosis (necrotic enteritis [NE]) was first recorded byBennetts (1930) in Australia,” thus these descriptions indicateda likely link to strain NCTC8503, a C. perfringens strain isolatedin 1930. We agree that no type D C. perfringens strains havebeen linked with poultry NE. Unfortunately, as NCTC8503 is ahistorical isolate with no detailed source information recorded,we have been unable to definitively confirm the isolate’s origin(Bennetts, 1930; Williams, 2005). However, we can reaffirmthat this isolate has not lost the epsilon toxin plasmid duringlaboratory passage as confirmed by both WGS (Figure 1), andmultiplex PCR toxinotyping (Baums et al., 2004; Kiu et al.,2017).The “extreme” level of pangenome (12.6% core genes) inC. perfringens has not been widely reported to date (McInerneyet al., 2017). However, as stated by the authors a previousstudy carried out in 2010 in Escherichia coli, which analysed53 genomes, indicated 11% core genes (Lukjancenko et al.,2010). Notably, although these 2 bacteria can colonise the GItract, they are fundamentally different regarding their oxygensensitivity and ability to form spores. As described by Lukjanenkoet al., analysis of 53 genomes (not 61 genomes as claimed)identified 1,472 core genes out of 13,296 genes in the pangenome(11%), which is lower than the 12.6% reported in our genomicC. perfringens analysis, although we note some differences in1https://www.phe-culturecollections.org.uk/approaches used. More recently a pangenome study based on228 E. coli genomes identified 23.8% core genes (2,722 coregenes in 11,401 gene families) (McNally et al., 2016), andusing web-based tool panX pangenome analysis2 and databases(Ding et al., 2018), based on the same cutoff as our studyindicated 13% of core genes, based on analysis of 307 E. coligenomes (as of May 2018). We reiterate that our “extreme”observation for spore-forming Gram-positive C. perfringens isa rare trait, and is therefore of interest to the wider researchcommunity. We agree with their statement “describing a speciesas having extreme variation depends very much to what it isbeing compared,” and furthermore we would emphasise thatprediction of genetic diversity will likely vary due to differentfactors including sampling bias, number of strains selected, andparameters used during informatics analysis, thus impactingdiversitymeasurements. In this study we comparedC. perfringenswith Clostridium difficile (30.3%, which has been reclassified asClostridioides difficile), a closer relative of C. perfringens, andStreptococcus pneumoniae (46.5%), Salmonella enterica (16%),and Klebsiella pneumoniae (26%) (Lawson et al., 2016; Kiu et al.,2017).We agree that there is currently no definitive pathogenicitylink with C. perfringens prophages, although previous studieshave indicated they enhance sporulation, which would beexpected to enhance transmission, and can be viewed as avirulence trait (Stewart and Johnson, 1977). Notably, in theclosely related pathogen C. difficile, bacteriophages are linked totoxin-secretion (Goh et al., 2005). Consequently, we speculatethat C. perfringens phages contribute to their virulence, whichcould be confirmed in future studies.We hope that our commentary provides clarification andcontext and we look forward to wider discussion with allinvestigators in the C. perfringens field.AUTHOR CONTRIBUTIONSRK and LH co-wrote the manuscript. RK performed thebioinformatics analysis.FUNDINGFunding by a Wellcome Trust Investigator Award(100974/C/13/Z), and the Biotechnology and Biological SciencesResearch Council (BBSRC); Institute Strategic Programme GutMicrobes and Health BB/R012490/1, and Institute StrategicProgramme Gut Health and Food Safety BB/J004529/1 to LH.RK is a recipient of University of East Anglia InternationalBursary (Faculty of Medical and Health Sciences).2http://pangenome.tuebingen.mpg.de/REFERENCESBaums, C. G., Schotte, U., Amtsberg, G., and Goethe, R. (2004). Diagnosticmultiplex PCR for toxin genotyping of Clostridium perfringens isolates. Vet.Microbiol. 100, 11–16. doi: 10.1016/S0378-1135(03)00126-3Bennetts, H. W. (1930). Bacillus welchii and bowel lesions. With special referenceto a case of coccidiosis. Aust. Vet. J. 6, 153–154.Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., et al.(2009). BLAST+: architecture and applications. BMC Bioinformatics 10:421.doi: 10.1186/1471-2105-10-421Frontiers in Microbiology | www.frontiersin.org 3 August 2018 | Volume 9 | Article 1857Kiu and Hall Genomic Analysis of Clostridium perfringensDing,W., Baumdicker, F., and Neher, R. A. (2018). panX: pan-genome analysis andexploration. Nucleic Acids Res. 46, e5. doi: 10.1093/nar/gkx977Goh, S., Chang, B. J., and Riley, T. V. (2005). Effect of phage infection ontoxin production by Clostridium difficile. J. Med. Microbiol. 54(Pt 2), 129–135.doi: 10.1099/jmm.0.45821-0Keyburn, A. L., Bannam, T. L., Moore, R. J., and Rood, J. I. (2010). NetB, a pore-forming toxin from necrotic enteritis strains of Clostridium perfringens. Toxins2, 1913–1927. doi: 10.3390/toxins2071913Keyburn, A. L., Boyce, J. D., Vaz, P., Bannam, T. L., Ford, M. E., Parker,D., et al. (2008). NetB, a new toxin that is associated with aviannecrotic enteritis caused by Clostridium perfringens. PLoS Pathog. 4:e26.doi: 10.1371/journal.ppat.0040026Kiu, R., Caim, S., Alexander, S., Pachori, P., and Hall, L. J. (2017). Probing genomicaspects of the multi-host pathogen Clostridium perfringens reveals significantpangenome diversity, and a diverse array of virulence factors. Front. Microbiol.8:2485. doi: 10.3389/fmicb.2017.02485Lawson, P. A., Citron, D. M., Tyrrell, K. L., and Finegold, S. M.(2016). Reclassification of Clostridium difficile as Clostridioidesdifficile (Hall and O’Toole 1935) Prevot 1938. Anaerobe 40, 95–99.doi: 10.1016/j.anaerobe.2016.06.008Lukjancenko, O., Wassenaar, T. M., and Ussery, D. W. (2010). Comparisonof 61 sequenced Escherichia coli genomes. Microb. Ecol. 60, 708–720.doi: 10.1007/s00248-010-9717-3McInerney, J. O., McNally, A., and O’Connell, M. J. (2017). Why prokaryotes havepangenomes. Nat. Microbiol. 2:17040. doi: 10.1038/nmicrobiol.2017.40McNally, A., Oren, Y., Kelly, D., Pascoe, B., Dunn, S., Sreecharan, T., et al.(2016). Combined analysis of variation in core, accessory and regulatorygenome regions provides a super-resolution view into the evolution ofbacterial populations. PLoS Genet. 12:e1006280. doi: 10.1371/journal.pgen.1006280Mehdizadeh Gohari, I., Parreira, V. R., Nowell, V. J., Nicholson, V. M.,Oliphant, K., and Prescott, J. F. (2015). A novel pore-forming toxin in typeA Clostridium perfringens is associated with both fatal canine hemorrhagicgastroenteritis and fatal foal necrotizing enterocolitis. PLoS ONE 10:e0122684.doi: 10.1371/journal.pone.0122684Nairn, M. E., and Bamford, V. W. (1967). Necrotic enteritis of broiler chickens inwestern Australia. Aust. Vet. J. 43, 49–54.Pearson, W. R. (2013). An introduction to sequence similarity(“homology”) searching. Curr. Protoc. Bioinformatics Chapter 3, Unit3 1.doi: 10.1002/0471250953.bi0301s42Rood, J. I., Keyburn, A. L., and Moore, R. J. (2016). NetB andnecrotic enteritis: the hole movable story. Avian Pathol. 45, 295–301.doi: 10.1080/03079457.2016.1158781Stewart, A. W., and Johnson, M. G. (1977). Increased numbers of heat-resistantspores produced by two strains of Clostridium perfringens bearing temperatephage s9. J. Gen. Microbiol. 103, 45–50. doi: 10.1099/00221287-103-1-45Williams, R. B. (2005). Intercurrent coccidiosis and necrotic enteritis of chickens:rational, integrated disease management by maintenance of gut integrity.AvianPathol. 34, 159–180. doi: 10.1080/03079450500112195Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.Copyright © 2018 Kiu and Hall. This is an open-access article distributed under theterms of the Creative Commons Attribution License (CC BY). The use, distributionor reproduction in other forums is permitted, provided the original author(s) andthe copyright owner(s) are credited and that the original publication in this journalis cited, in accordance with accepted academic practice. No use, distribution orreproduction is permitted which does not comply with these terms.Frontiers in Microbiology | www.frontiersin.org 4 August 2018 | Volume 9 | Article 1857

Response: Commentary: Probing genomic aspects of the multi-host pathogen Clostridium perfringens reveals significant pangenome diversity, and a diverse array of virulence factors

GlimmerGlass Volume 58 Number 06 (1998)

GlimmerGlass Volume 60 Number 01 (2000)

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Response: Commentary: Probing Genomic Aspects of the Multi-Host Pathogen Clostridium perfringens Reveals Significant Pangenome Diversity, and a Diverse Array of Virulence Factors

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