1887

Abstract

is highly polymorphic, and some strains are much more likely to cause disease than others. Biofilm formation can help bacteria to survive antibiotic treatment, immune attack and other stresses, promoting persistent infection.

We hypothesized that isolates from patients with more severe associated disease would be better at forming biofilms than isolates from patients with less severe disease.

We initially aimed to determine whether or not the biofilm-forming ability of isolates was associated with disease in the UK-based patients from whom the bacteria were isolated.

Biofilm-forming ability of isolates was determined using a crystal violet assay on glass coverslips. The complete genome sequence of strain 444A was generated by hybrid assembly of Nanopore MinION and Illumina MiSeq data.

Although we found no associations between biofilm-forming ability of and disease severity in patients, we discovered that strain 444A had particularly high biofilm-forming ability. This strain had been isolated from a patient with gastric ulcer disease and moderate to severe scores for induced histopathology. Analysis of the genome of the high biofilm-forming strain 444A revealed that it possesses numerous biofilm- and virulence-associated genes and a small cryptic plasmid encoding a type II toxin–antitoxin system.

There is substantial variation in biofilm-forming ability in but this was not significantly associated with disease severity in our study. We identified and characterized an interesting strain with high biofilm-forming ability, including generation and analysis of the complete genome.

Funding
This study was supported by the:
  • Government of the State of Kuwait
    • Principle Award Recipient: LolwahAlsharaf
  • NIHR Nottingham Biomedical Research Centre
    • Principle Award Recipient: KarenRobinson
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.001710
2023-06-09
2024-06-23
Loading full text...

Full text loading...

/deliver/fulltext/jmm/72/6/jmm001710.html?itemId=/content/journal/jmm/10.1099/jmm.0.001710&mimeType=html&fmt=ahah

References

  1. Atherton JC, Blaser MJ. Coadaptation of Helicobacter pylori and humans: ancient history, modern implications. J Clin Invest 2009; 119:2475–2487 [View Article] [PubMed]
    [Google Scholar]
  2. Blaser MJ, Berg DE. Helicobacter pylori genetic diversity and risk of human disease. J Clin Invest 2001; 107:767–773 [View Article] [PubMed]
    [Google Scholar]
  3. Hanafiah A, Lopes BS. Genetic diversity and virulence characteristics of Helicobacter pylori isolates in different human ethnic groups. Infect Genet Evol 2020; 78:104135 [View Article] [PubMed]
    [Google Scholar]
  4. Odenbreit S, Püls J, Sedlmaier B, Gerland E, Fischer W et al. Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science 2000; 287:1497–1500 [View Article] [PubMed]
    [Google Scholar]
  5. Atherton JC, Cao P, Peek RM, Tummuru MKR, Blaser MJ et al. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. J Biol Chemist 1995; 270:17771–17777 [View Article]
    [Google Scholar]
  6. Rhead JL, Letley DP, Mohammadi M, Hussein N, Mohagheghi MA et al. A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate region, is associated with gastric cancer. Gastroenterology 2007; 133:926–936 [View Article] [PubMed]
    [Google Scholar]
  7. Donlan RM. Biofilms: microbial life on surfaces. Emerg Infect Dis 2002; 8:881–890 [View Article] [PubMed]
    [Google Scholar]
  8. Carron M, Tran V, Sugawa C, Coticchia J. Identification of Helicobacter pylori biofilms in human gastric mucosa. J Gastrointest Surg 2006; 10:712–717 [View Article]
    [Google Scholar]
  9. Windham IH, Servetas SL, Whitmire JM, Pletzer D, Hancock REW et al. Helicobacter pylori biofilm formation is differentially affected by common culture conditions, and proteins play a central role in the biofilm matrix. Appl Environ Microbiol 2018; 84:e00391-18 [View Article] [PubMed]
    [Google Scholar]
  10. Cole SP, Harwood J, Lee R, She R, Guiney DG. Characterization of monospecies biofilm formation by Helicobacter pylori. J Bacteriol 2004; 186:3124–3132 [View Article] [PubMed]
    [Google Scholar]
  11. Hathroubi S, Zerebinski J, Clarke A, Ottemann KM. Helicobacter pylori biofilm confers antibiotic tolerance in part via A protein-dependent mechanism. Antibiotics 2020; 9:355 [View Article] [PubMed]
    [Google Scholar]
  12. Cai Y, Wang C, Chen Z, Xu Z, Li H et al. Transporters HP0939, HP0497, and HP0471 participate in intrinsic multidrug resistance and biofilm formation in Helicobacter pylori by enhancing drug efflux. Helicobacter 2020; 25:e12715 [View Article] [PubMed]
    [Google Scholar]
  13. Fauzia KA, Miftahussurur M, Syam AF, Waskito LA, Doohan D et al. Biofilm formation and antibiotic resistance phenotype of Helicobacter pylori clinical isolates. Toxins 2020; 12:473 [View Article] [PubMed]
    [Google Scholar]
  14. Yonezawa H, Osaki T, Hojo F, Kamiya S. Effect of Helicobacter pylori biofilm formation on susceptibility to amoxicillin, metronidazole and clarithromycin. Microb Pathog 2019; 132:100–108 [View Article] [PubMed]
    [Google Scholar]
  15. Krzyżek P, Migdał P, Grande R, Gościniak G. Biofilm formation of Helicobacter pylori in both static and microfluidic conditions is associated with resistance to clarithromycin. Front Cell Infect Microbiol 2022; 12:868905 [View Article] [PubMed]
    [Google Scholar]
  16. Yonezawa H, Osaki T, Kurata S, Fukuda M, Kawakami H et al. Outer membrane vesicles of Helicobacter pylori TK1402 are involved in biofilm formation. BMC Microbiol 2009; 9:197 [View Article] [PubMed]
    [Google Scholar]
  17. Hathroubi S, Hu S, Ottemann KM. Genetic requirements and transcriptomics of Helicobacter pylori biofilm formation on abiotic and biotic surfaces. NPJ Biofilms Microbiomes 2020; 6:56 [View Article] [PubMed]
    [Google Scholar]
  18. Wong EHJ, Ng CG, Chua EG, Tay ACY, Peters F et al. Comparative genomics revealed multiple Helicobacter pylori genes associated with biofilm formation in vitro. PLoS One 2016; 11:e0166835 [View Article] [PubMed]
    [Google Scholar]
  19. Attaran B, Falsafi T. Identification of factors associated with biofilm formation ability in the clinical isolates of Helicobacter pylori. Iran J Biotechnol 2017; 15:58–66 [View Article] [PubMed]
    [Google Scholar]
  20. Leunk RD, Johnson PT, David BC, Kraft WG, Morgan DR. Cytotoxic activity in broth-culture filtrates of Campylobacter pylori. J Med Microbiol 1988; 26:93–99 [View Article] [PubMed]
    [Google Scholar]
  21. Porechop RR. Porechop; 2017 https://github.com/rrwick/Porechop
  22. Lin Y, Yuan J, Kolmogorov M, Shen MW, Chaisson M et al. Assembly of long error-prone reads using de Bruijn graphs. Proc Natl Acad Sci 2016; 113:E8396–E8405 [View Article] [PubMed]
    [Google Scholar]
  23. Hunt M, Silva ND, Otto TD, Parkhill J, Keane JA et al. Circlator: automated circularization of genome assemblies using long sequencing reads. Genome Biol 2015; 16:294 [View Article] [PubMed]
    [Google Scholar]
  24. Wick RR, Schultz MB, Zobel J, Holt KE. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics 2015; 31:3350–3352 [View Article] [PubMed]
    [Google Scholar]
  25. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018; 34:i884–i890 [View Article] [PubMed]
    [Google Scholar]
  26. Vaser R, Sović I, Nagarajan N, Šikić M. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res 2017; 27:737–746 [View Article] [PubMed]
    [Google Scholar]
  27. 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] [PubMed]
    [Google Scholar]
  28. Pedersen BS, Quinlan AR, Hancock J. Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics 2018; 34:867–868 [View Article]
    [Google Scholar]
  29. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014; 30:2068–2069 [View Article]
    [Google Scholar]
  30. Wick RR, Judd LM, Gorrie CL, Holt KE, Phillippy AM. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 2017; 13:e1005595 [View Article]
    [Google Scholar]
  31. Jolley KA, Maiden MCJ. BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics 2010; 11:595 [View Article] [PubMed]
    [Google Scholar]
  32. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. Bioinformatics 2013; 29:1072–1075 [View Article] [PubMed]
    [Google Scholar]
  33. van Tonder AJ, Mistry S, Bray JE, Hill DMC, Cody AJ et al. Defining the estimated core genome of bacterial populations using a Bayesian decision model. PLoS Comput Biol 2014; 10:e1003788 [View Article]
    [Google Scholar]
  34. Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 2011; 12:402 [View Article] [PubMed]
    [Google Scholar]
  35. Gressmann H, Linz B, Ghai R, Pleissner K-P, Schlapbach R et al. Gain and loss of multiple genes during the evolution of Helicobacter pylori. PLoS Genet 2005; 1:e43 [View Article]
    [Google Scholar]
  36. Tomb JF, White O, Kerlavage AR, Clayton RA, Sutton GG et al. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 1997; 388:539–547 [View Article] [PubMed]
    [Google Scholar]
  37. Mannion A, Dzink-Fox J, Shen Z, Piazuelo MB, Wilson KT et al. Helicobacter pylori antimicrobial resistance and gene variants in high- and low-gastric-cancer-risk populations. J Clin Microbiol 2021; 59:e03203-20 [View Article] [PubMed]
    [Google Scholar]
  38. Gutiérrez-Escobar AJ, Velapatiño B, Borda V, Rabkin CS, Tarazona-Santos E et al. Identification of new Helicobacter pylori subpopulations in Native Americans and Mestizos From Peru. Front Microbiol 2020; 11:601839 [View Article] [PubMed]
    [Google Scholar]
  39. Binh TT, Suzuki R, Kwon DH, Yamaoka Y. Complete genome sequence of a metronidazole-resistant Helicobacter pylori strain. Genome Announc 2015; 3:e00051-15 [View Article] [PubMed]
    [Google Scholar]
  40. Valiente-Mullor C, Beamud B, Ansari I, Francés-Cuesta C, García-González N et al. One is not enough: on the effects of reference genome for the mapping and subsequent analyses of short-reads. PLoS Comput Biol 2021; 17:e1008678 [View Article]
    [Google Scholar]
  41. Tedersoo L, Albertsen M, Anslan S, Callahan B. Perspectives and benefits of high-throughput long-read sequencing in microbial ecology. Appl Environ Microbiol 2021; 87:e0062621 [View Article] [PubMed]
    [Google Scholar]
  42. Delahaye C, Nicolas J. Sequencing DNA with nanopores: troubles and biases. PLoS One 2021; 16:e0257521 [View Article] [PubMed]
    [Google Scholar]
  43. Nguyen-Hoang TP, Nguyen Hoa T, Nguyen TH, Baker S, Rahman M et al. Complete genome sequence of Helicobacter pylori strain GD63, isolated from a vietnamese patient with a gastric ulcer. Microbiol Resour Announc 2019; 8:e01412-18 [View Article] [PubMed]
    [Google Scholar]
  44. Takahashi M, Matsumoto Y, Ujihara T, Maeda H, Hanazaki K et al. Complete genome sequence of Helicobacter pylori strain 3401, a suitable host for bacteriophages KHP30 and KHP40. Microbiol Resour Announc 2021; 10:e0064721 [View Article] [PubMed]
    [Google Scholar]
  45. Mwangi C, Njoroge S, Tshibangu-Kabamba E, Moloo Z, Rajula A et al. Whole genome sequencing reveals virulence potentials of Helicobacter pylori strain KE21 isolated from a Kenyan patient with gastric signet ring cell carcinoma. Toxins 2020; 12:556 [View Article] [PubMed]
    [Google Scholar]
  46. Jin F, Yang H, Rasko D. Complete genome sequence of Helicobacter pylori strain 3192, isolated from a Chinese patient with chronic nonatrophic gastritis. Microbiol Resour Announc 2022; 11:e0038222 [View Article] [PubMed]
    [Google Scholar]
  47. Shao C, Sun Y, Wang N, Yu H, Zhou Y et al. Changes of proteome components of Helicobacter pylori biofilms induced by serum starvation. Mol Med Rep 2013; 8:1761–1766 [View Article]
    [Google Scholar]
  48. Hathroubi S, Zerebinski J, Ottemann KM. Helicobacter pylori biofilm involves a multigene stress-biased response, including a structural role for Flagella. mBio 2018; 9:e01973-18 [View Article] [PubMed]
    [Google Scholar]
  49. Wong EHJ, Ng CG, Goh KL, Vadivelu J, Ho B et al. Metabolomic analysis of low and high biofilm-forming Helicobacter pylori strains. Sci Rep 2018; 8:1409 [View Article] [PubMed]
    [Google Scholar]
  50. Servetas SL, Doster RS, Kim A, Windham IH, Cha J-H et al. ArsRS-dependent regulation of homB contributes to Helicobacter pylori biofilm formation. Front Microbiol 2018; 9: [View Article]
    [Google Scholar]
  51. Baltrus DA, Amieva MR, Covacci A, Lowe TM, Merrell DS et al. The complete genome sequence of Helicobacter pylori strain G27. J Bacteriol 2009; 191:447–448 [View Article] [PubMed]
    [Google Scholar]
  52. Hofreuter D, Haas R. Characterization of two cryptic Helicobacter pylori plasmids: a putative source for horizontal gene transfer and gene shuffling. J Bacteriol 2002; 184:2755–2766 [View Article] [PubMed]
    [Google Scholar]
  53. Han K-D, Matsuura A, Ahn H-C, Kwon A-R, Min Y-H et al. Functional identification of toxin-antitoxin molecules from Helicobacter pylori 26695 and structural elucidation of the molecular interactions. J Biol Chem 2011; 286:4842–4853 [View Article] [PubMed]
    [Google Scholar]
  54. Loris R, Garcia-Pino A. Disorder- and dynamics-based regulatory mechanisms in toxin-antitoxin modules. Chem Rev 2014; 114:6933–6947 [View Article] [PubMed]
    [Google Scholar]
  55. Leplae R, Geeraerts D, Hallez R, Guglielmini J, Drèze P et al. Diversity of bacterial type II toxin-antitoxin systems: a comprehensive search and functional analysis of novel families. Nucleic Acids Res 2011; 39:5513–5525 [View Article] [PubMed]
    [Google Scholar]
  56. Pathak C, Im H, Yang Y-J, Yoon H-J, Kim H-M et al. Crystal structure of apo and copper bound HP0894 toxin from Helicobacter pylori 26695 and insight into mRNase activity. Biochim Biophys Acta 2013; 1834:2579–2590 [View Article] [PubMed]
    [Google Scholar]
  57. Cárdenas-Mondragón MG, Ares MA, Panunzi LG, Pacheco S, Camorlinga-Ponce M et al. Transcriptional profiling of type II toxin-antitoxin genes of Helicobacter pylori under different environmental conditions: identification of HP0967-HP0968 system. Front Microbiol 2016; 7:1872 [View Article] [PubMed]
    [Google Scholar]
  58. Yonezawa H, Osaki T, Fukutomi T, Hanawa T, Kurata S et al. Diversification of the AlpB outer membrane protein of Helicobacter pylori affects biofilm formation and cellular adhesion. J Bacteriol 2017; 199:e00729-16 [View Article] [PubMed]
    [Google Scholar]
  59. Ge X, Cai Y, Chen Z, Gao S, Geng X et al. Bifunctional enzyme SpoT is involved in biofilm formation of Helicobacter pylori with multidrug resistance by upregulating efflux pump Hp1174 (gluP). Antimicrob Agents Chemother 2018; 62:e00957-18 [View Article] [PubMed]
    [Google Scholar]
  60. Zhao Y, Cai Y, Chen Z, Li H, Xu Z et al. SpoT-mediated NapA upregulation promotes oxidative stress-induced Helicobacter pylori biofilm formation and confers multidrug resistance. Antimicrob Agents Chemother 2023; 65:e00152-21 [View Article] [PubMed]
    [Google Scholar]
  61. Yang F-L, Hassanbhai AM, Chen H-Y, Huang Z-Y, Lin T-L et al. Proteomannans in biofilm of Helicobacter pylori ATCC 43504. Helicobacter 2011; 16:89–98 [View Article] [PubMed]
    [Google Scholar]
  62. Anderson JK, Huang JY, Wreden C, Sweeney EG, Goers J et al. Chemorepulsion from the quorum signal autoinducer-2 promotes Helicobacter pylori biofilm dispersal. mBio 2015; 6:e00379 [View Article] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.001710
Loading
/content/journal/jmm/10.1099/jmm.0.001710
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
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