1887

Abstract

is an important opportunistic pathogen associated with severe invasive disease in humans. Hypervirulent , which are with several acquired virulence determinants such as the siderophore aerobactin and others, are more prominent in countries in South and South-East Asia compared to European countries. This pathotype is capable of causing liver abscesses in immunocompetent persons in the community. has not been extensively studied in non-human niches. In the present study, isolated from caecal samples (=299) from healthy fattening pigs in Norway were characterized with regard to population structure and virulence determinants. These data were compared to data from a previous study on from healthy pigs in Thailand. Lastly, an in-depth plasmid study on with aerobactin was performed. Culturing and whole-genome sequencing was applied to detect, confirm and characterize isolates. Phylogenetic analysis described the evolutionary relationship and diversity of the isolates, while virulence determinants and sequence types were detected with Kleborate. Long-read sequencing was applied to obtain the complete sequence of virulence plasmids harbouring aerobactin. A total of 48.8 % of the investigated Norwegian pig caecal samples (=299) were positive for . Acquired virulence determinants were detected in 72.6 % of the isolates, the most prominent being aerobactin (69.2 %), all of which were . In contrast, only 4.6 % of the isolates from Thailand harboured aerobactin. The aerobactin operon was located on potentially conjugative IncFIB/FII plasmids of varying sizes in isolates from both countries. A putative, highly conserved composite transposon with a mean length of 16.2 kb flanked by truncated IS-family IS-group insertion sequences was detected on these plasmids, harbouring the aerobactin operon as well as several genes that may confer increased fitness in mammalian hosts. This putative composite transposon was also detected in plasmids harboured by from several countries and sources, such as human clinical samples. The high occurrence of harbouring aerobactin in Norwegian pigs, taken together with international data, suggest that pigs are a reservoir for with . Truncation of the flanking ISKpn78-element suggest that the putative composite transposon has been permanently integrated into the plasmid, and that it is no longer mobilizable.

Funding
This study was supported by the:
  • Trond Mohn stiftelse (Award TMS2019TMT03)
    • Principle Award Recipient: MarianneSunde
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.000960
2023-02-23
2024-03-03
Loading full text...

Full text loading...

/deliver/fulltext/mgen/9/2/mgen000960.html?itemId=/content/journal/mgen/10.1099/mgen.0.000960&mimeType=html&fmt=ahah

References

  1. WHO Critically Important Antimicrobials for Human Medicine Geneva: World Health Organization; 2017 p 41
    [Google Scholar]
  2. Wyres KL, Lam MMC, Holt KE. Population genomics of Klebsiella pneumoniae. Nat Rev Microbiol 2020; 18:344–359 [View Article]
    [Google Scholar]
  3. Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci 2015; 112:E3574–E3581 [View Article]
    [Google Scholar]
  4. Lam MMC, Wyres KL, Wick RR, Judd LM, Fostervold A et al. Convergence of virulence and MDR in a single plasmid vector in MDR Klebsiella pneumoniae ST15. J Antimicrob Chemother 2019; 74:1218–1222 [View Article] [PubMed]
    [Google Scholar]
  5. Turton JF, Payne Z, Coward A, Hopkins KL, Turton JA et al. Virulence genes in isolates of Klebsiella pneumoniae from the UK during 2016, including among carbapenemase gene-positive hypervirulent K1-ST23 and “non-hypervirulent” types ST147, ST15 and ST383. J Med Microbiol 2018; 67:118–128 [View Article] [PubMed]
    [Google Scholar]
  6. Dong N, Lin D, Zhang R, Chan EW-C, Chen S. Carriage of blaKPC-2 by a virulence plasmid in hypervirulent Klebsiella pneumoniae. J Antimicrob Chemother 2018; 73:3317–3321 [View Article]
    [Google Scholar]
  7. Arakawa Y, Ohta M, Wacharotayankun R, Mori M, Kido N et al. Biosynthesis of Klebsiella K2 capsular polysaccharide in Escherichia coli HB101 requires the functions of rmpA and the chromosomal cps gene cluster of the virulent strain Klebsiella pneumoniae Chedid (O1:K2). Infect Immun 1991; 59:2043–2050 [View Article]
    [Google Scholar]
  8. Russo TA, Olson R, MacDonald U, Beanan J, Davidson BA. Aerobactin, but not yersiniabactin, salmochelin, or enterobactin, enables the growth/survival of hypervirulent (hypermucoviscous) Klebsiella pneumoniae ex vivo and in vivo. Infect Immun 2015; 83:3325–3333 [View Article]
    [Google Scholar]
  9. Russo TA, Marr CM. Hypervirulent Klebsiella pneumoniae. Clin Microbiol Rev 2019; 32:e00001-19 [View Article] [PubMed]
    [Google Scholar]
  10. Russo TA, Olson R, Macdonald U, Metzger D, Maltese LM et al. Aerobactin mediates virulence and accounts for increased siderophore production under iron-limiting conditions by hypervirulent (hypermucoviscous) Klebsiella pneumoniae. Infect Immun 2014; 82:2356–2367 [View Article]
    [Google Scholar]
  11. Lam MMC, Wyres KL, Judd LM, Wick RR, Jenney A et al. Tracking key virulence loci encoding aerobactin and salmochelin siderophore synthesis in Klebsiella pneumoniae. Genome Med 2018; 10:77 [View Article] [PubMed]
    [Google Scholar]
  12. Klaper K, Hammerl JA, Rau J, Pfeifer Y, Werner G. Genome-based analysis of Klebsiella spp. isolates from animals and food products in Germany, 2013–2017. Pathogens 2021; 10:573 [View Article]
    [Google Scholar]
  13. Wyres KL, Nguyen TNT, Lam MMC, Judd LM, van Vinh Chau N et al. Genomic surveillance for hypervirulence and multi-drug resistance in invasive Klebsiella pneumoniae from South and Southeast Asia. Genome Med 2020; 12:11 [View Article] [PubMed]
    [Google Scholar]
  14. Bidewell CA, Williamson SM, Rogers J, Tang Y, Ellis RJ et al. Emergence of Klebsiella pneumoniae subspecies pneumoniae as a cause of septicaemia in pigs in England. PLoS One 2018; 13:e0191958 [View Article]
    [Google Scholar]
  15. Thorpe HA, Booton R, Kallonen T, Gibbon MJ, Couto N et al. A large-scale genomic snapshot of Klebsiella spp. isolates in Northern Italy reveals limited transmission between clinical and non-clinical settings. Nat Microbiol 2022; 7:2054–2067 [View Article] [PubMed]
    [Google Scholar]
  16. Leangapichart T, Lunha K, Jiwakanon J, Angkititrakul S, Järhult JD et al. Characterization of Klebsiella pneumoniae complex isolates from pigs and humans in farms in Thailand: population genomic structure, antibiotic resistance and virulence genes. J Antimicrob Chemother 2021; 76:2012–2016 [View Article]
    [Google Scholar]
  17. NORM/NORM-VET Usage of Antimicrobial Agents and Occurrence of Antimicrobial Resistance in Norway. Tromsø, Oslo: Norwegian Veterinary Institute/University Hospital of North Norway; 2020 https://www.vetinst.no/overvaking/antibiotikaresistens-norm-vet
  18. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 2017; 13:e1005595 [View Article]
    [Google Scholar]
  19. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. Bioinformatics 2013; 29:1072–1075 [View Article]
    [Google Scholar]
  20. Lam MMC, Wick RR, Watts SC, Cerdeira LT, Wyres KL et al. A genomic surveillance framework and genotyping tool for Klebsiella pneumoniae and its related species complex. Nat Commun 2021; 12:4188 [View Article]
    [Google Scholar]
  21. Kaspersen H, Fiskebeck EZ. ALPPACA - A tooL for prokaryotic phylogeny and clustering analysis. JOSS 2022; 7:4677 [View Article]
    [Google Scholar]
  22. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014; 30:2068–2069 [View Article] [PubMed]
    [Google Scholar]
  23. Tonkin-Hill G, MacAlasdair N, Ruis C, Weimann A, Horesh G et al. Producing polished prokaryotic pangenomes with the Panaroo pipeline. Genome Biol 2020; 21:180 [View Article] [PubMed]
    [Google Scholar]
  24. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 2013; 30:772–780 [View Article] [PubMed]
    [Google Scholar]
  25. Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 2015; 32:268–274 [View Article]
    [Google Scholar]
  26. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 2017; 14:587–589 [View Article] [PubMed]
    [Google Scholar]
  27. Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol 2018; 35:518–522 [View Article]
    [Google Scholar]
  28. R Core Team R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2021 https://www.R-project.org/
  29. Yu G. Using ggtree to visualize data on tree-like structures. Curr Protoc Bioinformatics 2020; 69:e96 [View Article]
    [Google Scholar]
  30. Xu S, Dai Z, Guo P, Fu X, Liu S et al. ggtreeExtra: compact visualization of richly annotated phylogenetic data. Mol Biol Evol 2021; 38:4039–4042 [View Article]
    [Google Scholar]
  31. De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 2018; 34:2666–2669 [View Article]
    [Google Scholar]
  32. Wick RR. Filtlong. In: Filtlong [Internet]. 2021 [cited 17 Nov 2021]; 2021 https://github.com/rrwick/Filtlong
  33. Wick RR, Judd LM, Cerdeira LT, Hawkey J, Méric G et al. Trycycler: consensus long-read assemblies for bacterial genomes. Genome Biol 2021; 22:266 [View Article] [PubMed]
    [Google Scholar]
  34. Wick RR, Holt KE. Benchmarking of long-read assemblers for prokaryote whole genome sequencing. F1000Res 2019; 8:2138 [View Article] [PubMed]
    [Google Scholar]
  35. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 2019; 37:540–546 [View Article] [PubMed]
    [Google Scholar]
  36. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 2014; 9:e112963 [View Article]
    [Google Scholar]
  37. Robertson J, Nash JHE. MOB-suite: software tools for clustering, reconstruction and typing of plasmids from draft assemblies. Microb Genom 2018; 4:e000206 [View Article]
    [Google Scholar]
  38. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 2012; 67:2640–2644 [View Article] [PubMed]
    [Google Scholar]
  39. Joensen KG, Scheutz F, Lund O, Hasman H, Kaas RS et al. Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli. J Clin Microbiol 2014; 52:1501–1510 [View Article] [PubMed]
    [Google Scholar]
  40. Carattoli A, Zankari E, García-Fernández A, Voldby Larsen M, Lund O et al. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother 2014; 58:3895–3903 [View Article] [PubMed]
    [Google Scholar]
  41. Schwengers O, Jelonek L, Dieckmann MA, Beyvers S, Blom J et al. Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microb Genom 2021; 7:000685 [View Article]
    [Google Scholar]
  42. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 2018; 34:3094–3100 [View Article] [PubMed]
    [Google Scholar]
  43. Johansson MHK, Bortolaia V, Tansirichaiya S, Aarestrup FM, Roberts AP et al. Detection of mobile genetic elements associated with antibiotic resistance in Salmonella enterica using a newly developed web tool: MobileElementFinder. J Antimicrob Chemother 2021; 76:101–109 [View Article] [PubMed]
    [Google Scholar]
  44. Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res 2006; 34:D32–D36 [View Article]
    [Google Scholar]
  45. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009; 25:1754–1760 [View Article] [PubMed]
    [Google Scholar]
  46. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009; 25:2078–2079 [View Article]
    [Google Scholar]
  47. Treangen TJ, Ondov BD, Koren S, Phillippy AM. The harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes. Genome Biol 2014; 15:524 [View Article]
    [Google Scholar]
  48. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J et al. BLAST+: architecture and applications. BMC Bioinformatics 2009; 10:421 [View Article]
    [Google Scholar]
  49. Callewaert L, Van Herreweghe JM, Vanderkelen L, Leysen S, Voet A et al. Guards of the great wall: bacterial lysozyme inhibitors. Trends Microbiol 2012; 20:501–510 [View Article]
    [Google Scholar]
  50. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Biol Evol 2018; 35:1547–1549 [View Article]
    [Google Scholar]
  51. Franklin-Alming F, Kaspersen H, Hetland MAK, Bakksjø R-J, Nesse LL et al. Exploring Klebsiella pneumoniae in healthy poultry reveals high genetic diversity, good biofilm-forming abilities and higher prevalence in turkeys than broilers. Front Microbiol 2021; 12:11 [View Article]
    [Google Scholar]
  52. Raffelsberger N, Hetland MAK, Svendsen K, Småbrekke L, Löhr IH et al. Gastrointestinal carriage of Klebsiella pneumoniae in a general adult population: a cross-sectional study of risk factors and bacterial genomic diversity. Gut Microbes 2021; 13:1939599 [View Article]
    [Google Scholar]
  53. Huynh B-T, Passet V, Rakotondrasoa A, Diallo T, Kerleguer A et al. Klebsiella pneumoniae carriage in low-income countries: antimicrobial resistance, genomic diversity and risk factors. Gut Microbes 2020; 11:1287–1299 [View Article]
    [Google Scholar]
  54. Fostervold A, Hetland MAK, Bakksjø R, Bernhoff E, Holt KE et al. A nationwide genomic study of clinical Klebsiella pneumoniae in Norway 2001–15: introduction and spread of ESBLs facilitated by clonal groups CG15 and CG307. J Antimicrob Chemother 2022; 77:665–674 [View Article]
    [Google Scholar]
  55. Tian D, Wang M, Zhou Y, Hu D, Ou H-Y et al. Genetic diversity and evolution of the virulence plasmids encoding aerobactin and salmochelin in Klebsiella pneumoniae. Virulence 2021; 12:1323–1333 [View Article]
    [Google Scholar]
  56. Tian D, Wang W, Li M, Chen W, Zhou Y et al. Acquisition of the conjugative virulence plasmid from a CG23 hypervirulent Klebsiella pneumoniae strain enhances bacterial virulence. Front Cell Infect Microbiol 2021; 11:752011 [View Article]
    [Google Scholar]
  57. Yang X, Chan E-C, Zhang R, Chen S. A conjugative plasmid that augments virulence in Klebsiella pneumoniae. Nat Microbiol 2019; 4:2039–2043 [View Article]
    [Google Scholar]
  58. Genuini M, Bidet P, Benoist J-F, Schlemmer D, Lemaitre C et al. ShiF acts as an auxiliary factor of aerobactin secretion in meningitis Escherichia coli strain S88. BMC Microbiol 2019; 19:298 [View Article] [PubMed]
    [Google Scholar]
  59. Callewaert L, Aertsen A, Deckers D, Vanoirbeek KGA, Vanderkelen L et al. A new family of lysozyme inhibitors contributing to lysozyme tolerance in gram-negative bacteria. PLoS Pathog 2008; 4:e1000019 [View Article] [PubMed]
    [Google Scholar]
  60. Callewaert L, Michiels CW. Lysozymes in the animal kingdom. J Biosci 2010; 35:127–160 [View Article]
    [Google Scholar]
  61. Hengge R. Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol 2009; 7:263–273 [View Article]
    [Google Scholar]
  62. Struve C, Bojer M, Krogfelt KA. Characterization of Klebsiella pneumoniae type 1 fimbriae by detection of phase variation during colonization and infection and impact on virulence. Infect Immun 2008; 76:4055–4065 [View Article]
    [Google Scholar]
  63. Teplyakov A, Obmolova G, Toedt J, Galperin MY, Gilliland GL. Crystal structure of the bacterial YhcH protein indicates a role in sialic acid catabolism. J Bacteriol 2005; 187:5520–5527 [View Article] [PubMed]
    [Google Scholar]
  64. Vallese F, Percudani R, Fischer W, Zanotti G. The crystal structure of Helicobacter pylori HP1029 highlights the functional diversity of the sialic acid-related DUF386 family. FEBS J 2015; 282:3311–3322 [View Article] [PubMed]
    [Google Scholar]
  65. Severi E, Hood DW, Thomas GH. Sialic acid utilization by bacterial pathogens. Microbiology 2007; 153:2817–2822 [View Article]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/mgen/10.1099/mgen.0.000960
Loading
/content/journal/mgen/10.1099/mgen.0.000960
Loading

Data & Media loading...

Supplements

Supplementary material 1

EXCEL

Supplementary material 2

EXCEL

Supplementary material 3

EXCEL

Supplementary material 4

EXCEL
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error