Horizontal Gene Transfer Among Neisseria Species and Humans

  • S. SánchezEmail author
  • T. de Miguel
  • T. G. Villa
  • A. R. Gorringe
  • I. M. Feavers


The genus Neisseria is included among those organisms containing a large number of foreign or exogenous genes in its genome. In most cases, the incorporation of these foreign genes is the consequence of recent events of horizontal gene transfer (HGT) occurring between different species of Neisseria but also with other bacterial genera such as Haemophilus influenzae and even with its exclusive host, the human. In this chapter, we review the transformation process as the main mechanism of HGT in Neisseria and its role in the acquisition of virulence factors and antibiotic resistance markers, mainly from commensal to pathogen Neisseria spp. In addition, we review evidence of gene exchange between both pathogenic Neisseria spp. and human, showing that HGT can occur between mammal’s host and their associated bacteria.


Neisseria spp. Horizontal gene transfer Transformation Neisseria meningitidis Neisseria gonorrhoeae Antibiotic resistance 


  1. Aas FE, Løvold C, Koomey M (2002a) An inhibitor of DNA binding and uptake events dictates the proficiency of genetic transformation in Neisseria gonorrhoeae: mechanism of action and links to type IV pilus expression. Mol Microbiol 46(5):1441–1450PubMedCrossRefGoogle Scholar
  2. Aas FE et al (2002b) Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression. Mol Microbiol 46(3):749–760. PubMedCrossRefGoogle Scholar
  3. Aho EL et al (2005) Neisserial pilin genes display extensive interspecies diversity. FEMS Microbiol Lett 249(2):327–334. PubMedCrossRefGoogle Scholar
  4. Ambur OH, Frye SA, Tønjum T (2007) New functional identity for the DNA uptake sequence in transformation and its presence in transcriptional terminators. J Bacteriol 189(5):2077–2085. PubMedCrossRefGoogle Scholar
  5. Andersen B et al (1993) Neisseria weaveri sp. nov., formerly CDC Group M-5, a Gram-negative bacterium associated with dog bite wounds. J Clin Microbiol 31(9):2456–2466PubMedPubMedCentralGoogle Scholar
  6. Anderson MT, Seifert HS (2011a) Neisseria gonorrhoeae and humans perform an evolutionary LINE dance. Mob Genet Elem 1(1):85–87. CrossRefGoogle Scholar
  7. Anderson MT, Seifert HS (2011b) Opportunity and means: horizontal gene transfer from the human host to a bacterial pathogen. MBio 2(1):e00005–e00011. PubMedPubMedCentralCrossRefGoogle Scholar
  8. Assalkhou R et al (2007) The outer membrane secretin PilQ from Neisseria meningitidis binds DNA. Microbiology 153(5):1593–1603. PubMedPubMedCentralCrossRefGoogle Scholar
  9. Bart D, van der Ende (1999) Antigenic variation of the class I outer membrane protein in hyperendemic Neisseria meningitidis strains in the Netherlands. Infect Immun 67(8):3842–3846PubMedPubMedCentralGoogle Scholar
  10. Beernink PT, Granoff DM (2009) The modular architecture of meningococcal factor H-binding protein. Microbiology 155(Pt 9):2873–2883. PubMedPubMedCentralCrossRefGoogle Scholar
  11. Benam AV et al (2011) Structure-function relationships of the competence lipoprotein ComL and SSB in meningococcal transformation. Microbiology 157(Pt 5):1329–1342. PubMedPubMedCentralCrossRefGoogle Scholar
  12. Berry J-L et al (2013) Functional analysis of the interdependence between DNA uptake sequence and its cognate ComP receptor during natural transformation in Neisseria species. PLoS Genet 9(12):e1004014. PubMedPubMedCentralCrossRefGoogle Scholar
  13. Beyene GT et al (2017) Comparative proteomic analysis of Neisseria meningitidis wildtype and dprA null mutant strains links DNA processing to pilus biogenesis. BMC Microbiol 17(1):1–18. CrossRefGoogle Scholar
  14. Biswas GD et al (1977) Factors affecting genetic transformation of Neisseria gonorrhoeae. J Bacteriol 129(2):983–992PubMedPubMedCentralGoogle Scholar
  15. Biswas GD, Thompson SA, Sparling PF (1989) Gene transfer in Neisseria gonorrhoeae. Clin Microbiol Rev 2:S24–S28PubMedPubMedCentralCrossRefGoogle Scholar
  16. Bovre K, Holten E (1970) Neisseria elongata sp. nov., a rod-shaped member of the genus Neisseria. Re-evaluation of cell shape as a criterion in classification. J Gen Microbiol 60(1):67–75. PubMedCrossRefGoogle Scholar
  17. Bowler LD et al (1994) Interspecies recombination between the penA genes of Neisseria meningitidis and commensal Neisseria species during the emergence of penicillin resistance in N. meningitidis: natural events and laboratory simulation. J Bacteriol 176(2):333–337PubMedPubMedCentralCrossRefGoogle Scholar
  18. Brynildsrud OB et al (2018) Acquisition of virulence genes by a carrier strain gave rise to the ongoing epidemics of meningococcal disease in West Africa. Proc Natl Acad Sci U S A 115(21):5510–5515. PubMedPubMedCentralCrossRefGoogle Scholar
  19. Budroni S et al (2011) Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous recombination. Proc Natl Acad Sci U S A 108(11):4494–4499. PubMedPubMedCentralCrossRefGoogle Scholar
  20. Callaghan MM et al (2017) Secretion of chromosomal DNA by the Neisseria gonorrhoeae type IV secretion system. Mol Microbiol 413:323–345. CrossRefGoogle Scholar
  21. Cartwright KA et al (1987) The Stonehouse survey: nasopharyngeal carriage of meningococci and Neisseria lactamica. Epidemiol Infect 99(3):591–601PubMedPubMedCentralCrossRefGoogle Scholar
  22. Caugant DA, Maiden MCJ (2009) Meningococcal carriage and disease--population biology and evolution. Vaccine 27(2):B64–B70. PubMedPubMedCentralCrossRefGoogle Scholar
  23. Caugant DA et al (1994) Asymptomatic carriage of Neisseria meningitidis in a randomly sampled population. J Clin Microbiol 32(2):323–330PubMedPubMedCentralGoogle Scholar
  24. Cehovin A et al (2013) Specific DNA recognition mediated by a type IV pilin. Proc Natl Acad Sci 110(8):3065–3070. PubMedCrossRefGoogle Scholar
  25. Chaussee MS, Hill SA (1998) Formation of single-stranded DNA during DNA transformation of Neisseria gonorrhoeae. J Bacteriol 180(19):5117–5122PubMedPubMedCentralGoogle Scholar
  26. Chen I, Dubnau D (2004) DNA uptake during bacterial transformation. Nat Rev Microbiol 2(3):241–249. PubMedCrossRefGoogle Scholar
  27. Chen I, Gotschlich EC (2001) ComE, a competence protein from Neisseria gonorrhoeae with DNA-binding activity. J Bacteriol 183(10):3160–3168. PubMedPubMedCentralCrossRefGoogle Scholar
  28. Claus H et al (2000) Differential distribution of novel restriction-modification systems in clonal lineages of Neisseria meningitidis. J Bacteriol 182(5):1296–1303PubMedPubMedCentralCrossRefGoogle Scholar
  29. Claus H et al (2005) Genetic analysis of meningococci carried by children and young adults. J Infect Dis 191(8):1263–1271. PubMedCrossRefGoogle Scholar
  30. Clemence MEA, Maiden MCJ, Harrison OB (2018) Characterization of capsule genes in non-pathogenic Neisseria species. Microbial Genomics 4:1–12. CrossRefGoogle Scholar
  31. Collins RF et al (2003) Three-dimensional structure of the Neisseria meningitidis secretin PilQ determined from negative-stain transmission electron microscopy. J Bacteriol 185(8):2611–2617PubMedPubMedCentralCrossRefGoogle Scholar
  32. Cury GCG et al (2014) Inflammatory response of Haemophilus influenzae biotype aegyptius causing Brazilian Purpuric fever. Braz J Microbiol 45(4):1449–1454PubMedCrossRefGoogle Scholar
  33. Davidsen T et al (2004) Biased distribution of DNA uptake sequences towards genome maintenance genes. Nucleic Acids Res 32(3):1050–1058. PubMedPubMedCentralCrossRefGoogle Scholar
  34. Davis J et al (2001) Evolution of an autotransporter: domain shuffling and lateral transfer from pathogenic Haemophilus to Neisseria. J Bacteriol 183(15):4626–4635. PubMedPubMedCentralCrossRefGoogle Scholar
  35. Dietrich M et al (2011) Activation of NF-kappaB by Neisseria gonorrhoeae is associated with microcolony formation and type IV pilus retraction. Cell Microbiol 13(8):1168–1182. PubMedCrossRefGoogle Scholar
  36. Doherty AJ, Serpell LC, Ponting CP (1996) The helix-hairpin-helix DNA-binding motif: a structural basis for non-sequence-specific recognition of DNA. Nucleic Acids Res 24(13):2488–2497PubMedPubMedCentralCrossRefGoogle Scholar
  37. Domingues S, Nielsen KM (2017) Membrane vesicles and horizontal gene transfer in prokaryotes. Curr Opin Microbiol 38:16–21. PubMedCrossRefGoogle Scholar
  38. Dorward DW, Garon CF, Judd RC (1989) Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae. J Bacteriol 171(5):2499–2505. PubMedPubMedCentralCrossRefGoogle Scholar
  39. Duffin PM, Seifert HS (2012) Genetic transformation of Neisseria gonorrhoeae shows a strand preference. FEMS Microbiol Lett 334(1):44–48. PubMedPubMedCentralCrossRefGoogle Scholar
  40. Dyet KH, Martin DR (2005) Sequence variation in the porB gene from B:P1.4 Meningococci causing New Zealand’s epidemic. J Clin Microbiol 43(2):838–842. PubMedPubMedCentralCrossRefGoogle Scholar
  41. Elkins C et al (1991) Species-specific uptake of DNA by gonococci is mediated by a 10-base-pair sequence. J Bacteriol 173(12):3911–3913PubMedPubMedCentralCrossRefGoogle Scholar
  42. Facius D, Meyer TF (1993) A novel determinant (comA) essential for natural transformation competence in Neisseria gonorrhoeae and the effect of a comA defect on pilin variation. Mol Microbiol 10(4):699–712PubMedCrossRefGoogle Scholar
  43. Feavers IM et al (1992) Role of horizontal genetic exchange in the antigenic variation of the class 1 outer membrane protein of Neisseria meningitidis. Mol Microbiol 6(4):489–495PubMedCrossRefGoogle Scholar
  44. Fermer C et al (1995) Sulfonamide resistance in Neisseria meningitidis as defined by site-directed mutagenesis could have its origin in other species. J Bacteriol 177(16):4669–4675PubMedPubMedCentralCrossRefGoogle Scholar
  45. Frosch M, Meyer TF (1992) Transformation-mediated exchange of virulence determinants by co-cultivation of pathogenic Neisseriae. FEMS Microbiol Lett 100(1–3):345–349PubMedCrossRefGoogle Scholar
  46. Frye SA et al (2013) Dialects of the DNA uptake sequence in Neisseriaceae. PLoS Genet 9(4):e1003458. PubMedPubMedCentralCrossRefGoogle Scholar
  47. Frye SA et al (2015) The inner membrane protein PilG interacts with DNA and the secretin PilQ in transformation. PLoS One 10(8):e0134954. PubMedPubMedCentralCrossRefGoogle Scholar
  48. Fussenegger M et al (1997) Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae--a review. Gene 192(1):125–134PubMedCrossRefGoogle Scholar
  49. Gangel H et al (2014) Concerted spatio-temporal dynamics of imported DNA and ComE DNA uptake protein during gonococcal transformation. PLoS Pathog 10(4):e1004043. PubMedPubMedCentralCrossRefGoogle Scholar
  50. Goodman SD, Scoccat JJ (1988) Skrip Lakonan Arab TAC 501. 85(September):6982–6986Google Scholar
  51. Goodman SD et al (2006) Mu-like Prophage in serogroup B Neisseria meningitidis coding for surface-exposed antigens. J Clin Microbiol 85(4):2580–2588. CrossRefGoogle Scholar
  52. Hamilton HL, Dillard JP (2006) Natural transformation of Neisseria gonorrhoeae: from DNA donation to homologous recombination. Mol Microbiol 59(2):376–385. PubMedCrossRefGoogle Scholar
  53. Han XY, Hong T, Falsen E (2006) Neisseria bacilliformis sp. nov. isolated from human infections. J Clin Microbiol 44(2):474–479. PubMedPubMedCentralCrossRefGoogle Scholar
  54. Harrison OB, Maiden MC, Rokbi B (2008) Distribution of transferrin binding protein B gene (tbpB) variants among Neisseria species. BMC Microbiol 8(1):66. PubMedPubMedCentralCrossRefGoogle Scholar
  55. Hepp C, Maier B (2016) Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism. Proc Natl Acad Sci U S A 113(44):12467–12472. PubMedPubMedCentralCrossRefGoogle Scholar
  56. Hobbs MM et al (1994) Microevolution within a clonal population of pathogenic bacteria: recombination, gene duplication and horizontal genetic exchange in the opa gene family of Neisseria meningitidis. Mol Microbiol 12(2):171–180PubMedCrossRefGoogle Scholar
  57. Holmes B et al (1993) Neisseria weaveri sp. nov. (formerly CDC group M-5), from dog bite wounds of humans. Int J Syst Bacteriol 43(4):687–693. PubMedCrossRefGoogle Scholar
  58. Hotopp JC et al (2006) Comparative genomics of Neisseria meningitidis: core genome, islands of horizontal transfer and pathogen-specific genes. Microbiology 152(Pt 12):3733–3749. CrossRefGoogle Scholar
  59. Hovland E et al (2017) DprA from Neisseria meningitidis: properties and role in natural competence for transformation. Microbiology 163(7):1016–1029. PubMedPubMedCentralCrossRefGoogle Scholar
  60. Joseph B et al (2011) Virulence evolution of the human pathogen Neisseria meningitidis by recombination in the core and accessory genome. PLoS One 6(4):e18441. PubMedPubMedCentralCrossRefGoogle Scholar
  61. Karch A, Vogel U, Claus H (2015) Role of penA polymorphisms for penicillin susceptibility in Neisseria lactamica and Neisseria meningitidis. Int J Med Microbiol 305(7):729–735. PubMedCrossRefGoogle Scholar
  62. Kłyż A, Piekarowicz A (2018) Phage proteins are expressed on the surface of Neisseria gonorrhoeae and are potential vaccine candidates. PLoS One 13(8):e0202437. PubMedPubMedCentralCrossRefGoogle Scholar
  63. Lauer P, Albertson NH, Koomey M (1993) Conservation of genes encoding components of a type IV pilus assembly/two-step protein export pathway in Neisseria gonorrhoeae. Mol Microbiol 8(2):357–368PubMedCrossRefGoogle Scholar
  64. Lewis DA (2010) The Gonococcus fights back: is this time a knock out? Sex Transm Infect 86(6):415–421. PubMedCrossRefGoogle Scholar
  65. Li M-S et al (2003) Identification and characterization of genomic loci unique to the Brazilian purpuric fever clonal group of H. influenzae biogroup aegyptius: functionality explored using meningococcal homology. Mol Microbiol 47(4):1101–1111PubMedCrossRefGoogle Scholar
  66. Linz B et al (2000) Frequent interspecific genetic exchange between commensal neisseriae and Neisseria meningitidis. Mol Microbiol 36(5):1049–1058. PubMedCrossRefGoogle Scholar
  67. Liu G, Tang CM, Exley RM (2015) Non-pathogenic Neisseria: members of an abundant, multi-habitat, diverse genus. Microbiology 161(7):1297–1312. PubMedCrossRefGoogle Scholar
  68. Lorenz MG, Wackernagel W (1994) Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev 58(3):563–602PubMedPubMedCentralGoogle Scholar
  69. Maiden MC et al (1998) Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A 95(6):3140–3145PubMedPubMedCentralCrossRefGoogle Scholar
  70. Marri PR et al (2010) Genome sequencing reveals widespread virulence gene exchange among human Neisseria species. PLoS One 5(7):e11835. PubMedPubMedCentralCrossRefGoogle Scholar
  71. Mehr IJ, Seifert HS (1998) Differential roles of homologous recombination pathways in Neisseria gonorrhoeae pilin antigenic variation, DNA transformation and DNA repair. Mol Microbiol 30(4):697–710. PubMedCrossRefGoogle Scholar
  72. Merz AJ, So M, Sheetz MP (2000) Pilus retraction powers bacterial twitching motility. Nature 407(6800):98–102. PubMedCrossRefGoogle Scholar
  73. Mongold JA (1992) DNA repair and the evolution of transformation in Haemophilus influenzae. Genetics 132(4):893–898PubMedPubMedCentralGoogle Scholar
  74. Mulhall RM et al (2016) Resolution of a protracted Serogroup B meningococcal outbreak with whole-genome sequencing shows interspecies genetic transfer. J Clin Microbiol 54(12):2891–2899. PubMedPubMedCentralCrossRefGoogle Scholar
  75. Obergfell KP, Seifert S (2015) Mobile DNA in the pathogenic Neisseria. In: Mobile DNA III. American Society of Microbiology, Washington, DC, pp 451–469. CrossRefGoogle Scholar
  76. Ohnishi M et al (2010) Spread of a chromosomal cefixime-resistant penA gene among different Neisseria gonorrhoeae lineages. Antimicrob Agents Chemother 54(3):1060–1067. PubMedCrossRefGoogle Scholar
  77. Pandey A, Cleary DW, Laver JR, Gorringe A, Deasy AM, Dale AP, Morris PD, Didelot X, Maiden MCJ, Read RC (2018) Microevolution of Neisseria lactamica during nasopharyngeal colonisation induced by controlled human infection. Nat Commun 9(1):4753PubMedPubMedCentralCrossRefGoogle Scholar
  78. Piekarowicz A et al (2014) Neisseria gonorrhoeae filamentous phage Ngo 6 is capable of infecting a variety of Gram-negative bacteria. J Virol 88(2):1002–1010. PubMedPubMedCentralCrossRefGoogle Scholar
  79. Qvarnstrom Y, Swedberg G (2006) Variations in gene organization and DNA uptake signal sequence in the folP region between commensal and pathogenic Neisseria species. BMC Microbiol 6:11. PubMedPubMedCentralCrossRefGoogle Scholar
  80. Rådström P et al (1992) Transformational exchanges in the dihydropteroate synthase gene of Neisseria meningitidis: a novel mechanism for acquisition of sulfonamide resistance. J Bacteriol 174(20):6386–6393PubMedPubMedCentralCrossRefGoogle Scholar
  81. Ramsey ME, Woodhams KL, Dillard JP (2011) The gonococcal genetic island and type IV secretion in the pathogenic Neisseria. Front Microbiol 2:1–9. CrossRefGoogle Scholar
  82. Roberts MC (1989) Plasmids of Neisseria gonorrhoeae and other Neisseria species. Clin Microbiol Rev 2:18–23. CrossRefGoogle Scholar
  83. Schoen C et al (2008) Whole-genome comparison of disease and carriage strains provides insights into virulence evolution in Neisseria meningitidis. Proc Natl Acad Sci U S A 105(9):3473–3478. PubMedPubMedCentralCrossRefGoogle Scholar
  84. Serino L, Virji M (2002) Genetic and functional analysis of the phosphorylcholine moiety of commensal Neisseria lipopolysaccharide. Mol Microbiol 43(2):437–448PubMedCrossRefGoogle Scholar
  85. Smith HO, Gwinn ML, Salzberg SL (1999) DNA uptake signal sequences in naturally transformable bacteria. Res Microbiol 150(9–10):603–616. PubMedCrossRefGoogle Scholar
  86. Sparling PF (1966) Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance. J Bacteriol 92(5):1364–1371PubMedPubMedCentralGoogle Scholar
  87. Sparling PF (1986) The roles of sexual and asexual gene transfer in emergence of antibiotic resistant gonococci. Trans Am Clin Climatol Assoc 97:60–68PubMedPubMedCentralGoogle Scholar
  88. Spratt BG et al (1992) Role of interspecies transfer of chromosomal genes in the evolution of penicillin resistance in pathogenic and commensal Neisseria species. J Mol Evol 34(2):115–125PubMedCrossRefGoogle Scholar
  89. Steinberg VI et al (1976) Isolation and characterization of a bacteriophage specific for Neisseria perflava. J Clin Microbiol 4(1):87–91PubMedPubMedCentralGoogle Scholar
  90. Stephens DS (1999) Uncloaking the meningococcus: dynamics of carriage and disease. Lancet 353(9157):941–942. PubMedCrossRefGoogle Scholar
  91. Swanson J (1973) Studies on gonococcus infection. IV. Pili: their role in attachment of gonococci to tissue culture cells. J Exp Med 137(3):571–589PubMedPubMedCentralCrossRefGoogle Scholar
  92. Thulin S et al (2006) Total variation in the penA gene of Neisseria meningitidis: correlation between susceptibility to beta-lactam antibiotics and penA gene heterogeneity. Antimicrob Agents Chemother 50(10):3317–3324. PubMedPubMedCentralCrossRefGoogle Scholar
  93. Unemo M, Shafer WM (2011) Antibiotic resistance in Neisseria gonorrhoeae: origin, evolution, and lessons learned for the future. Ann N Y Acad Sci 1230(1):E19–E28. PubMedPubMedCentralCrossRefGoogle Scholar
  94. Wang X et al (2015) Changes in the population structure of invasive Neisseria meningitidis in the United States after Quadrivalent meningococcal conjugate vaccine licensure. J Infect Dis 211(12):1887–1894. PubMedCrossRefGoogle Scholar
  95. Weinstein P (1991) The Australian bushfly (Musca vetustissima Walker) as a vector of Neisseria gonorrhoeae conjunctivitis. Med J Aust 155(10):717PubMedGoogle Scholar
  96. Whitchurch CB et al (1991) Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. Gene 101(1):33–44PubMedCrossRefGoogle Scholar
  97. WHO Guidelines for the Treatment of Neisseria gonorrhoeae (2016) World Health Organization, Geneva (no date)Google Scholar
  98. Wolfgang WJ et al (2011) Neisseria wadsworthii sp. nov. and Neisseria shayeganii sp. nov., isolated from clinical specimens. Int J Syst Evol Microbiol 61(1):91–98. PubMedCrossRefGoogle Scholar
  99. Xu Z et al (2015) Phylogenetic study of clonal complex (CC)198 capsule null locus (cnl) genomes: a distinctive group within the species Neisseria meningitidis. Infect Genet Evol 34:372–377. PubMedCrossRefGoogle Scholar
  100. Yero D et al (2010) Variation in the Neisseria meningitidis FadL-like protein: an evolutionary model for a relatively low-abundance surface antigen. Microbiology 156(12):3596–3608. PubMedCrossRefGoogle Scholar
  101. Zhang Y et al (2013) Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol Cell 50(4):488–503. PubMedPubMedCentralCrossRefGoogle Scholar
  102. Zhu P et al (2002) Genetic diversity of three lgt loci for biosynthesis of lipooligosaccharide (LOS) in Neisseria species. Microbiology 148(Pt 6):1833–1844. PubMedCrossRefGoogle Scholar

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© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • S. Sánchez
    • 1
    Email author
  • T. de Miguel
    • 1
  • T. G. Villa
    • 2
  • A. R. Gorringe
    • 3
  • I. M. Feavers
    • 4
  1. 1.Faculty of Pharmacy, Department of MicrobiologyUniversity of Santiago de CompostelaSantiago de CompostelaSpain
  2. 2.Faculty of Pharmacy, Department of MicrobiologyUniversity of Santiago de CompostelaSantiago de CompostelaSpain
  3. 3.Public Health EnglandSalisburyUK
  4. 4.National Institute for Biological Standards and ControlSouth Mimms, Potters BarUK

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