Development of a multi-locus typing scheme for an Enterobacteriaceae linear plasmid that mediates inter-species transfer of flagella

Autoři: James Robertson aff001;  Janet Lin aff001;  Amie Wren-Hedgus aff001;  Gitanjali Arya aff001;  Catherine Carrillo aff002;  John H. E. Nash aff003
Působiště autorů: National Microbiology Laboratory, Public Health Agency of Canada, Guelph, Ontario, Canada aff001;  Ottawa Laboratory (Carling), Canadian Food Inspection Agency, Ottawa, Ontario, Canada aff002;  National Microbiology Laboratory, Public Health Agency of Canada, Toronto, Ontario, Canada aff003
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0218638


Due to the public health importance of flagellar genes for typing, it is important to understand mechanisms that could alter their expression or presence. Phenotypic novelty in flagellar genes arise predominately through accumulation of mutations but horizontal transfer is known to occur. A linear plasmid termed pBSSB1 previously identified in Salmonella Typhi, was found to encode a flagellar operon that can mediate phase variation, which results in the rare z66 flagella phenotype. The identification and tracking of homologs of pBSSB1 is limited because it falls outside the normal replicon typing schemes for plasmids. Here we report the generation of nine new pBSSB1-family sequences using Illumina and Nanopore sequence data. Homologs of pBSSB1 were identified in 154 genomes representing 25 distinct serotypes from 67,758 Salmonella public genomes. Pangenome analysis of pBSSB1-family contigs was performed using roary and we identified three core genes amenable to a minimal pMLST scheme. Population structure analysis based on the newly developed pMLST scheme identified three major lineages representing 35 sequence types, and the distribution of these sequence types was found to span multiple serovars across the globe. This in silico pMLST scheme has shown utility in tracking and subtyping pBSSB1-family plasmids and it has been incorporated into the plasmid MLST database under the name “pBSSB1-family”.

Klíčová slova:

Flagella – Plasmid construction – Plasmids – Salmonella – Salmonella typhi – Sequence analysis – Sequence assembly tools – Sequence databases


1. Yoshida CE, Kruczkiewicz P, Laing CR, Lingohr EJ, Gannon VPJ, Nash JHE, et al. The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies. PLOS ONE. 2016 Jan 22;11(1):e0147101. doi: 10.1371/journal.pone.0147101 26800248

2. Franklin K, Lingohr EJ, Yoshida C, Anjum M, Bodrossy L, Clark CG, et al. Rapid Genoserotyping Tool for Classification of Salmonella Serovars▿. J Clin Microbiol. 2011 Aug;49(8):2954–65. doi: 10.1128/JCM.02347-10 21697324

3. Yoshida C, Gurnik S, Ahmad A, Blimkie T, Murphy SA, Kropinski AM, et al. Evaluation of molecular methods for the identification of Salmonella serovars. J Clin Microbiol. 2016 May 18;JCM.00262-16.

4. Broadbent SE, Davies MR, van der Woude MW. Phase variation controls expression of Salmonella lipopolysaccharide modification genes by a DNA methylation-dependent mechanism. Mol Microbiol. 2010 Jul;77(2):337–53. doi: 10.1111/j.1365-2958.2010.07203.x 20487280

5. Schnaitman CA, Klena JD. Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol Rev. 1993 Sep;57(3):655–82. 7504166

6. Silverman M, Zieg J, Hilmen M, Simon M. Phase variation in Salmonella: genetic analysis of a recombinational switch. Proc Natl Acad Sci U S A. 1979 Jan;76(1):391–5. doi: 10.1073/pnas.76.1.391 370828

7. Beltran P, Musser JM, Helmuth R, Farmer JJ, Frerichs WM, Wachsmuth IK, et al. Toward a population genetic analysis of Salmonella: genetic diversity and relationships among strains of serotypes S. choleraesuis, S. derby, S. dublin, S. enteritidis, S. heidelberg, S. infantis, S. newport, and S. typhimurium. Proc Natl Acad Sci. 1988 Oct 1;85(20):7753–7. doi: 10.1073/pnas.85.20.7753 3051004

8. Kropinski AM, Kovalyova IV, Billington SJ, Patrick AN, Butts BD, Guichard JA, et al. The Genome of ε15, a Serotype-Converting, Group E1 Salmonella enterica-Specific Bacteriophage. Virology. 2007 Dec 20;369(2):234–44. doi: 10.1016/j.virol.2007.07.027 17825342

9. Wright A. Mechanism of Conversion of the Salmonella O Antigen by Bacteriophage ε34. J Bacteriol. 1971 Mar;105(3):927–36. 5547996

10. Keenleyside WJ, Whitfield C. A Novel Pathway for O-Polysaccharide Biosynthesis in Salmonella enterica Serovar Borreze. J Biol Chem. 1996 Nov 8;271(45):28581–92. doi: 10.1074/jbc.271.45.28581 8910488

11. Rowe B, Hall ML, McCoy JH. Salmonella crossness—a new serotype containing a new comatic (O) antigen, 67. J Hyg (Lond). 1976 Dec;77(3):355–7.

12. Everest P, Wain J, Roberts M, Rook G, Dougan G. The molecular mechanisms of severe typhoid fever. Trends Microbiol. 2001 Jul;9(7):316–20. doi: 10.1016/s0966-842x(01)02067-4 11435104

13. Kidgell C, Reichard U, Wain J, Linz B, Torpdahl M, Dougan G, et al. Salmonella typhi, the causative agent of typhoid fever, is approximately 50,000 years old. Infect Genet Evol. 2002 Oct;2(1):39–45. doi: 10.1016/s1567-1348(02)00089-8 12797999

14. Pa G, Wh J, Hm M, L LM, R B. An unusual H antigen (Z66) in strains of Salmonella typhi. Ann Microbiol (Paris). 1980 1981;132(3):331–4.

15. Baker S, Hardy J, Sanderson KE, Quail M, Goodhead I, Kingsley RA, et al. A Novel Linear Plasmid Mediates Flagellar Variation in Salmonella Typhi. PLOS Pathog. 2007 May 11;3(5):e59. doi: 10.1371/journal.ppat.0030059 17500588

16. Yachison CA, Yoshida C, Robertson J, Nash JHE, Kruczkiewicz P, Taboada EN, et al. The Validation and Implications of Using Whole Genome Sequencing as a Replacement for Traditional Serotyping for a National Salmonella Reference Laboratory. Front Microbiol [Internet]. 2017 [cited 2017 Jul 17];8. Available from:

17. Nair S, Ashton P, Doumith M, Connell S, Painset A, Mwaigwisya S, et al. WGS for surveillance of antimicrobial resistance: a pilot study to detect the prevalence and mechanism of resistance to azithromycin in a UK population of non-typhoidal Salmonella. J Antimicrob Chemother. 2016 Sep 1;dkw318.

18. Nutrition C for FS and A. Whole Genome Sequencing (WGS) Program—GenomeTrakr Fast Facts [Internet]. [cited 2016 Nov 25]. Available from:

19. Wyres KL, Conway TC, Garg S, Queiroz C, Reumann M, Holt K, et al. WGS Analysis and Interpretation in Clinical and Public Health Microbiology Laboratories: What Are the Requirements and How Do Existing Tools Compare? Pathogens. 2014 Jun 11;3(2):437–58. doi: 10.3390/pathogens3020437 25437808

20. Zhang S, Yin Y, Jones MB, Zhang Z, Kaiser BLD, Dinsmore BA, et al. Salmonella Serotype Determination Utilizing High-Throughput Genome Sequencing Data. J Clin Microbiol. 2015 May 1;53(5):1685–92. doi: 10.1128/JCM.00323-15 25762776

21. Carattoli A, Zankari E, García-Fernández A, Voldby Larsen M, Lund O, Villa L, et al. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother. 2014 Jul;58(7):3895–903. doi: 10.1128/AAC.02412-14 24777092

22. Maiden MCJ, van Rensburg MJJ, Bray JE, Earle SG, Ford SA, Jolley KA, et al. MLST revisited: the gene-by-gene approach to bacterial genomics. Nat Rev Microbiol. 2013 Oct;11(10):728–36. doi: 10.1038/nrmicro3093 23979428

23. Achtman M, Wain J, Weill F-X, Nair S, Zhou Z, Sangal V, et al. Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica. PLOS Pathog. 2012 Jun 21;8(6):e1002776. doi: 10.1371/journal.ppat.1002776 22737074

24. Been M de, Pinholt M, Top J, Bletz S, Mellmann A, Schaik W van, et al. Core Genome Multilocus Sequence Typing Scheme for High-Resolution Typing of Enterococcus faecium. J Clin Microbiol. 2015 Dec 1;53(12):3788–97. doi: 10.1128/JCM.01946-15 26400782

25. Alikhan N-F, Zhou Z, Sergeant MJ, Achtman M. A genomic overview of the population structure of Salmonella. PLOS Genet. 2018 Apr 5;14(4):e1007261. doi: 10.1371/journal.pgen.1007261 29621240

26. Hancock SJ, Phan M-D, Peters KM, Forde BM, Chong TM, Yin W-F, et al. Identification of IncA/C plasmid replication and maintenance genes and development of a plasmid multilocus sequence typing scheme. Antimicrob Agents Chemother. 2017;61(2):e01740–16. doi: 10.1128/AAC.01740-16 27872077

27. García-Fernández A, Carattoli A. Plasmid double locus sequence typing for IncHI2 plasmids, a subtyping scheme for the characterization of IncHI2 plasmids carrying extended-spectrum beta-lactamase and quinolone resistance genes. J Antimicrob Chemother. 2010 Jun;65(6):1155–61. doi: 10.1093/jac/dkq101 20356905

28. García-Fernández A, Chiaretto G, Bertini A, Villa L, Fortini D, Ricci A, et al. Multilocus sequence typing of IncI1 plasmids carrying extended-spectrum beta-lactamases in Escherichia coli and Salmonella of human and animal origin. J Antimicrob Chemother. 2008 Jun;61(6):1229–33. doi: 10.1093/jac/dkn131 18367460

29. García-Fernández A, Villa L, Moodley A, Hasman H, Miriagou V, Guardabassi L, et al. Multilocus sequence typing of IncN plasmids. J Antimicrob Chemother. 2011 Sep;66(9):1987–91. doi: 10.1093/jac/dkr225 21653604

30. Zhang H, Zhu Y, Xie X, Wang M, Du H, Xu S, et al. Identification and Characterization of a Gene stp17 Located on the Linear Plasmid pBSSB1 as an Enhanced Gene of Growth and Motility in Salmonella enterica Serovar Typhi. Front Cell Infect Microbiol [Internet]. 2016 Oct 5;6. Available from:

31. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLOS Comput Biol. 2017 Jun 8;13(6):e1005595. doi: 10.1371/journal.pcbi.1005595 28594827

32. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017 Mar 15;gr.215087.116.

33. Robertson J, Yoshida C, Kruczkiewicz P, Nadon C, Nichani A, Taboada EN, et al. Comprehensive assessment of the quality of Salmonella whole genome sequence data available in public sequence databases using the Salmonella in silico Typing Resource (SISTR). Microb Genomics [Internet]. 2018 [cited 2018 Apr 3];4(2). Available from:

34. Robertson J, Nash JHE. MOB-suite: software tools for clustering, reconstruction and typing of plasmids from draft assemblies. Microb Genomics. 2018;4(8).

35. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014 Jul 15;30(14):2068–9. doi: 10.1093/bioinformatics/btu153 24642063

36. Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MTG, et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics. 2015 Nov 15;31(22):3691–3. doi: 10.1093/bioinformatics/btv421 26198102

37. Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002 Jul 15;30(14):3059–66. doi: 10.1093/nar/gkf436 12136088

38. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870–4. doi: 10.1093/molbev/msw054 27004904

39. Zhou Z, Alikhan N-F, Sergeant MJ, Luhmann N, Vaz C, Francisco AP, et al. GrapeTree: Visualization of core genomic relationships among 100,000 bacterial pathogens. Genome Res. 2018 Jul 26;gr.232397.117.

40. Li W, Godzik A. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 2006 Jul 1;22(13):1658–9. doi: 10.1093/bioinformatics/btl158 16731699

41. Sullivan MJ, Petty NK, Beatson SA. Easyfig: a genome comparison visualizer. Bioinformatics. 2011 Apr 1;27(7):1009–10. doi: 10.1093/bioinformatics/btr039 21278367

42. Jolley KA, Maiden MC. BIGSdb: Scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics. 2010 Dec 10;11(1):595.

43. Jolley KA, Bray JE, Maiden MCJ. Open-access bacterial population genomics: BIGSdb software, the website and their applications. Wellcome Open Res [Internet]. 2018 Sep 24 [cited 2019 Apr 9];3. Available from:

44. Guinée PA, Jansen WH, Maas HM, Le Minor L, Beaud R. An unusual H antigen (Z66) in strains of Salmonella typhi. Ann Microbiol (Paris). 1981 Jun;132(3):331–4.

Článek vyšel v časopise


2019 Číslo 11