Chapter 5 : Fast and High-Throughput Molecular Typing Methods

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in

Fast and High-Throughput Molecular Typing Methods, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555817121/9781555815424_Chap05-1.gif /docserver/preview/fulltext/10.1128/9781555817121/9781555815424_Chap05-2.gif


As our understanding of pathogens has expanded and the world’s food supply has become more interconnected, characterization and subtyping of foodborne pathogens have become critically important in monitoring specific foods, performing surveillance for foodborne pathogens, investigating foodborne outbreaks, and understanding the virulence properties of particular lineages or strains. This chapter provides an overview of the molecular subtyping methods most commonly employed, in particular focusing on those methods providing fast and high-throughput analysis of strains. The methods include pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable-number tandem repeat (VNTR) analysis, DNA microarrays, and mass spectrometry. The heterogeneity that is targeted in the methods falls into three general categories. First, single-nucleotide polymorphisms (SNPs) are single-base changes in one sequence in comparison to another, related sequence. SNPs typically arise through sporadic mutation, so they represent a means of measuring random genetic drift. A large number of SNPs between two sequences may indicate a more distant genetic relationship. Second, some regions of the genome are more likely to change than others; they provide a means of differentiating closely related strains. Examples include variable number tandem repeats and highly polymorphic genes such as some surface structure and virulence genes. Finally, the presence/absence of specific loci can be found in both distantly and closely related strains. The presence of additional sequences in a strain can result from the insertion of a bacteriophage in the chromosome, acquisition of a plasmid, or other horizontal transfer events.

Citation: Fields P, Fitzgerald C, McQuiston J. 2011. Fast and High-Throughput Molecular Typing Methods, p 81-92. In Hoorfar J (ed), Rapid Detection, Characterization, and Enumeration of Foodborne Pathogens. ASM Press, Washington, DC. doi: 10.1128/9781555817121.ch5
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


1. Afset, J. E.,, E. Anderssen,, G. Bruant,, J. Harel,, L. Wieler, and, K. Bergh. 2008. Phylogenetic backgrounds and virulence profiles of atypical enteropathogenic Escherichia coli strains from a case-control study using multilocus sequence typing and DNA microarray analysis. J. Clin. Microbiol. 46: 22802290.
2. Beranek, A.,, C. Mikula,, P. Rabold,, D. Arnhold,, C. Berghold,, I. Lederer,, F. Allerberger, and, C. Kornschober. 2009. Multiple-locus variable-number tandem repeat analysis for subtyping of Salmonella enterica subsp. enterica serovar Enteritidis. Int. J. Med. Microbiol. 299: 4351.
3. Beres, S. B.,, R. K. Carroll,, P. R. Shea,, I. Sitkiewicz,, J. C. Martinez-Gutierrez,, D. E. Low,, A. McGeer,, B. M. Willey,, K. Green,, G. J. Tyrrell,, T. D. Goldman,, M. Feldgarden,, B. W. Birren,, Y. Fofanov,, J. Boos,, W. D. Wheaton,, C. Honisch, and, J. M. Musser. 2010. Molecular complexity of successive bacterial epidemics deconvoluted by comparative pathogenomics. Proc. Natl. Acad. Sci. USA 107: 43714376.
4. Best, E. L.,, A. J. Fox,, J. A. Frost, and, F. J. Bolton. 2005. Real-time single-nucleotide polymorphism profiling using Taqman technology for rapid recognition of Campylobacter jejuni clonal complexes. J. Med. Microbiol. 54 (Pt. 10): 919925.
5. Bishop, C.,, T. Callman, and, J. Wain. 2010. Characterization of SNPs in Salmonella Paratyphi A using MALDI-TOF MS, p. 65–67. In P. Colin and, G. Clement (ed.), International Symposium on Salmonella and Salmonellosis. Zoopole Development-ISPAIA, Saint-Malo, France.
6. Bruant, G.,, C. Maynard,, S. Bekal,, I. Gaucher,, L. Masson,, R. Brousseau, and, J. Harel. 2006. Development and validation of an oligonucleotide microarray for detection of multiple virulence and antimicrobial resistance genes in Escherichia coli. Appl. Environ. Microbiol. 72: 37803784.
7. Cho, S.,, T. S. Whittam,, D. J. Boxrud,, J. M. Bartkus,, S. C. Rankin,, M. J. Wilkins,, P. Somsel,, F. P. Downes,, K. A. Musser,, T. P. Root,, L. D. Warnick,, M. Wiedmann, and, A. M. Saeed. 2010. Use of multiple-locus variable number tandem repeat analysis and phage typing for subtyping of Salmonella Enteritidis from sporadic human cases in the United States. J. Appl. Microbiol. 108: 859867.
8. Clark, C. G.,, L. Bryden,, W. R. Cuff,, P. L. Johnson,, F. Jamieson,, B. Ciebin, and, G. Wang. 2005. Use of the Oxford multilocus sequence typing protocol and sequencing of the flagellin short variable region to characterize isolates from a large outbreak of waterborne Campylobacter sp. strains in Walkerton, Ontario, Canada. J. Clin. Microbiol. 43: 20802091.
9. Cody, A. J.,, M. J. Maiden, and, K. E. Dingle. 2009. Genetic diversity and stability of the porA allele as a genetic marker in human Campylobacter infection. Microbiology 155 (Pt. 12): 41454154.
10. Davis, M. A.,, K. N. Baker,, D. R. Call,, L. D. Warnick,, Y. Soyer,, M. Wiedmann,, Y. Grohn,, P. L. McDonough,, D. D. Hancock, and, T. E. Besser. 2009. Multilocus variable-number tandem-repeat method for typing Salmonella enterica serovar Newport. J. Clin. Microbiol. 47: 19341938.
11. de Haan, C. P.,, R. Kivisto,, M. Hakkinen,, H. Rautelin, and, M. L. Hanninen. 2010. Decreasing trend of overlapping MLST genotypes between human and chicken Campylobacter jejuni isolates over a decade in Finland. Appl. Environ. Microbiol. 76: 52285236.
12. Dunbar, S. A. 2006. Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection. Clin. Chim. Acta 363: 7182.
13. Fakhr, M. K.,, L. K. Nolan, and, C. M. Logue. 2005. Multilocus sequence typing lacks the discriminatory ability of pulsed-field gel electrophoresis for typing Salmonella enterica serovar Typhimurium. J. Clin. Microbiol. 43: 22152219.
14. Fitzgerald, C.,, M. Collins,, S. van Duyne,, M. Mikoleit,, T. Brown, and, P. Fields. 2007. Multiplex, bead-based suspension array for molecular determination of common Salmonella serogroups. J. Clin. Microbiol. 45: 33233334.
15. Frye, J. G.,, R. L. Lindsey,, G. Rondeau,, S. Porwollik,, F. Long,, M. McClelland,, C. R. Jackson,, M. D. Englen,, R. J. Meinersmann,, M. E. Berrang,, J. A. Davis,, J. B. Barrett,, J. B. Turpin,, S. N. Thitaram, and, P. J. Fedorka-Cray. 2010. Development of a DNA microarray to detect antimicrobial resistance genes identified in the National Center for Biotechnology Information database. Microb. Drug Resist. 16: 919.
16. Garaizar, J.,, A. Rementeria, and, S. Porwollik. 2006. DNA microarray technology: a new tool for the epidemiological typing of bacterial pathogens? FEMS Immunol. Med. Microbiol. 47: 178189.
17. Gerner-Smidt, P.,, K. Hise,, J. Kincaid,, S. Hunter,, S. Rolando,, E. Hyytia-Trees,, E. M. Ribot, and, B. Swaminathan. 2006. PulseNet USA: a five-year update. Foodborne Pathog. Dis. 3: 919.
18. Gilmour, M. W.,, M. Graham,, G. Van Domselaar,, S. Tyler,, H. Kent,, K. M. Trout-Yakel,, O. Larios,, V. Allen,, B. Lee, and, C. Nadon. 2010. High-throughput genome sequencing of two Listeria monocytogenes clinical isolates during a large foodborne outbreak. BMC Genomics 11: 120.
19. Grønlund, H.,, L. Riber,, H. Vigre,, C. Löfström,, L. Folling,, S. Huehn,, B. Malorny,, P. Rådström,, K. Rudi, and, J. Hoorfar. 2010. Microarray-based genotyping of Salmonella: inter-laboratory evaluation of reproducibility and standardization potential. Int. J. Food Microbiol. Epub ahead of print. doi:10.1016/j.ijfoodmicro.2010. 08.007.
20. Hall, T. A.,, R. Sampath,, L. B. Blyn,, R. Ranken,, C. Ivy,, R. Melton,, H. Matthews,, N. White,, F. Li,, V. Harpin,, D. J. Ecker,, L. K. McDougal,, B. Limbago,, T. Ross,, D. M. Wolk,, V. Wysocki, and, K. C. Carroll. 2009. Rapid molecular genotyping and clonal complex assignment of Staphylococcus aureus isolates by PCR coupled to electrospray ionization-mass spectrometry. J. Clin. Microbiol. 47: 17331741.
21. Hamelin, K.,, G. Bruant,, A. El-Shaarawi,, S. Hill,, T. A. Edge,, J. Fairbrother,, J. Harel,, C. Maynard,, L. Masson, and, R. Brousseau. 2007. Occurrence of virulence and antimicrobial resistance genes in Escherichia coli isolates from different aquatic ecosystems within the St. Clair River and Detroit River areas. Appl. Environ. Microbiol. 73: 477484.
22. Honisch, C.,, Y. Chen,, C. Mortimer,, C. Arnold,, O. Schmidt,, D. van den Boom,, C. R. Cantor,, H. N. Shah, and, S. E. Gharbia. 2007. Automated comparative sequence analysis by base-specific cleavage and mass spectrometry for nucleic acid-based microbial typing. Proc. Natl. Acad. Sci. USA 104: 1064910654.
23. Honisch, C.,, M. Mosko,, C. Arnold,, S. E. Gharbia,, R. Diel, and, S. Niemann. 2010. Replacing reverse line blot hybridization spoligotyping of the Mycobacterium tuberculosis complex. J. Clin. Microbiol. 48: 15201526.
24. Huehn, S.,, C. Bunge,, E. Junker,, R. Helmuth, and, B. Malorny. 2009a. Poultry-associated Salmonella enterica subsp. enterica serovar 4,12:d:- reveals high clonality and a distinct pathogenicity gene repertoire. Appl. Environ. Microbiol. 75: 10111020.
25. Huehn, S.,, R. Helmuth,, C. Bunge,, B. Guerra,, E. Junker,, R. H. Davies,, P. Wattiau,, W. van Pelt, and, B. Malorny. 2009b. Characterization of pathogenic and resistant genome repertoire reveals two clonal lines in Salmonella enterica subsp. enterica serovar Paratyphi B (+)-tartrate positive. Foodborne Pathog. Dis. 6: 431443.
26. Huehn, S.,, and B. Malorny. 2009. DNA microarray for molecular epidemiology of Salmonella. Methods Mol. Biol. 551: 249285.
27. Hyytia-Trees, E.,, P. Lafon,, P. Vauterin, and, E. M. Ribot. 2010. Multilaboratory validation study of standardized multiple-locus variable-number tandem repeat analysis protocol for Shiga toxin-producing Escherichia coli O157: a novel approach to normalize fragment size data between capillary electrophoresis platforms. Foodborne Pathog. Dis. 7: 129136.
28. Hyytia-Trees, E.,, S. C. Smole,, P. A. Fields,, B. Swaminathan, and, E. M. Ribot. 2006. Second generation subtyping: a proposed PulseNet protocol for multiple-locus variable-number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 (STEC O157). Foodborne Pathog. Dis. 3: 118131.
29. Kerouanton, A.,, M. Marault,, R. Lailler,, F. X. Weill,, C. Feurer,, E. Espie, and, A. Brisabois. 2007. Pulsed-field gel electrophoresis subtyping database for foodborne Salmonella enterica serotype discrimination. Foodborne Pathog. Dis. 4: 293303.
30. Kotetishvili, M.,, O. C. Stine,, A. Kreger,, J. G. Morris, Jr., and, A. Sulakvelidze. 2002. Multilocus sequence typing for characterization of clinical and environmental Salmonella strains. J. Clin. Microbiol. 40: 16261635.
31. Lewis, T.,, N. J. Loman,, L. Bingle,, P. Jumaa,, G. M. Weinstock,, D. Mortiboy, and, M. J. Pallen. 2010. High-throughput whole-genome sequencing to dissect the epidemiology of Acinetobacter baumannii isolates from a hospital outbreak. J. Hosp. Infect. 75: 3741.
32. Lindstedt, B. A. 2005. Multiple-locus variable number tandem repeats analysis for genetic fingerprinting of pathogenic bacteria. Electrophoresis 26: 25672582.
33. Lindstedt, B. A.,, W. Tham,, M. L. Danielsson-Tham,, T. Vardund,, S. Helmersson, and, G. Kapperud. 2008. Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing. J. Microbiol. Methods 72: 141148.
34. Litrup, E.,, M. Torpdahl,, B. Malorny,, S. Huehn,, M. Helms,, H. Christensen, and, E. M. Nielsen. 2010. DNA microarray analysis of Salmonella sero-type Typhimurium strains causing different symptoms of disease. BMC Microbiol. 10: 96.
35. Maiden, M. C. 2006. Multilocus sequence typing of bacteria. Annu. Rev. Microbiol. 60: 561588.
36. Malorny, B.,, E. Junker, and, R. Helmuth. 2008. Multilocus variable-number tandem repeat analysis for outbreak studies of Salmonella enterica serotype Enteritidis. BMC Microbiol. 8: 84.
37. Mandrell, R. E.,, L. A. Harden,, A. Bates,, W. G. Miller,, W. F. Haddon, and, C. K. Fagerquist. 2005. Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Appl. Environ. Microbiol. 71: 62926307.
38. McQuiston, J. R.,, R. J. Waters,, B. Dinsmore,, M. Mikoleit, and, P. I. Fields. 15 December 2010. Molecular determination of H-antigens of Salmonella using a microsphere-based liquid array. J. Clin. Microbiol. (Epub ahead of print.)
39. Mellmann, A.,, F. Bimet,, C. Bizet,, A. D. Borovskaya,, R. R. Drake,, U. Eigner,, A. M. Fahr,, Y. He,, E. N. Ilina,, M. Kostrzewa,, T. Maier,, L. Mancinellie,, W. Moussaoui,, G. Prévost,, L. Putignani,, C. L. Seachord,, Y. W. Tang, and, D. Harmsen. 2009. High interlaboratory reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based species identification of nonfermenting bacteria. J. Clin. Microbiol. 47: 37323734.
40. Miko, A.,, B. A. Lindstedt,, L. T. Brandal,, I. Lobersli, and, L. Beutin. 2010. Evaluation of multiple-locus variable number of tandem-repeats analysis (MLVA) as a method for identification of clonal groups among enteropathogenic, enterohaemorrhagic and avirulent Escherichia coli O26 strains. FEMS Microbiol. Lett. 303: 137146.
41. Miller, M. B.,, and Y. W. Tang. 2009. Basic concepts of microarrays and potential applications in clinical microbiology. Clin. Microbiol. Rev. 22: 611633.
42. Murphy, M.,, D. Corcoran,, J. F. Buckley,, M. O’Mahony,, P. Whyte, and, S. Fanning. 2007. Development and application of multiple-locus variable number of tandem repeat analysis (MLVA) to subtype a collection of Listeria monocytogenes. Int. J. Food Microbiol. 115: 187194.
43. Ribot, E. M.,, M. A. Fair,, R. Gautom,, D. N. Cameron,, S. B. Hunter,, B. Swaminathan, and, T. J. Barrett. 2006. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog. Dis. 3: 5967.
44. Ross, I. L.,, and M. W. Heuzenroeder. 2005. Discrimination within phenotypically closely related definitive types of Salmonella enterica serovar Typhimurium by the multiple amplification of phage locus typing technique. J. Clin. Microbiol. 43: 16041611.
45. Ross, I. L.,, and M. W. Heuzenroeder. 2009. A comparison of two PCR-based typing methods with pulsed-field gel electrophoresis in Salmonella enterica serovar Enteritidis. Int. J. Med. Microbiol. 299: 410420.
46. Sauer, S.,, and M. Kliem. 2010. Mass spectrometry tools for the classification and identification of bacteria. Nat. Rev. Microbiol. 8: 7482.
47. Sheppard, S. K.,, J. F. Dallas,, M. MacRae,, N. D. McCarthy,, E. L. Sproston,, F. J. Gormley,, N. J. Strachan,, I. D. Ogden,, M. C. Maiden, and, K. J. Forbes. 2009. Campylobacter genotypes from food animals, environmental sources and clinical disease in Scotland 2005/6. Int. J. Food Microbiol. 134: 96103.
48. Sopwith, W.,, A. Birtles,, M. Matthews,, A. Fox,, S. Gee,, S. James,, J. Kempster,, M. Painter,, V. Edwards-Jones,, K. Osborn,, M. Regan,, Q. Syed, and, E. Bolton. 2010. Investigation of food and environmental exposures relating to the epidemiology of Campylobacter coli in humans in Northwest England. Appl. Environ. Microbiol. 76: 129135.
49. Soyer, Y.,, S. D. Alcaine,, D. J. Schoonmaker-Bopp,, T. P. Root,, L. D. Warnick,, P. L. McDonough,, N. B. Dumas,, Y. T. Grohn, and, M. Wiedmann. 2010. Pulsed-field gel electrophoresis diversity of human and bovine clinical Salmonella isolates. Foodborne Pathog. Dis. 7: 707717.
50. Sperry, K. E.,, S. Kathariou,, J. S. Edwards, and, L. A. Wolf. 2008. Multiple-locus variable-number tandem-repeat analysis as a tool for subtyping Listeria monocytogenes strains. J. Clin. Microbiol. 46: 14351450.
51. Sukhnanand, S.,, S. Alcaine,, L. D. Warnick,, W. L. Su,, J. Hof,, M. P. Craver,, P. McDonough,, K. J. Boor, and, M. Wiedmann. 2005. DNA sequence-based subtyping and evolutionary analysis of selected Salmonella enterica serotypes. J. Clin. Microbiol. 43: 36883698.
52. Swaminathan, B.,, P. Gerner-Smidt,, L. K. Ng,, S. Lukinmaa,, K. M. Kam,, S. Rolando,, E. P. Gutierrez, and, N. Binsztein. 2006. Building PulseNet International: an interconnected system of laboratory networks to facilitate timely public health recognition and response to foodborne disease outbreaks and emerging foodborne diseases. Foodborne Pathog. Dis. 3: 3650.
53. Torpdahl, M.,, M. N. Skov,, D. Sandvang, and, D. L. Baggesen. 2005. Genotypic characterization of Salmonella by multilocus sequence typing, pulsed-field gel electrophoresis and amplified fragment length polymorphism. J. Microbiol. Methods 63: 173184.
54. Torpdahl, M.,, G. Sorensen,, B. A. Lindstedt, and, E. M. Nielsen. 2007. Tandem repeat analysis for surveillance of human Salmonella Typhimurium infections. Emerg. Infect. Dis. 13: 388395.
55. Ward, T. J.,, T. F. Ducey,, T. Usgaard,, K. A. Dunn, and, J. P. Bielawski. 2008. Multilocus genotyping assays for single nucleotide polymorphism-based subtyping of Listeria monocytogenes isolates. Appl. Environ. Microbiol. 74: 76297642.
56. Wolk, D. M.,, L. B. Blyn,, T. A. Hall,, R. Sampath,, R. Ranken,, C. Ivy,, R. Melton,, H. Matthews,, N. White,, F. Li,, V. Harpin,, D. J. Ecker,, B. Limbago,, L. K. McDougal,, V. H. Wysocki,, M. Cai, and, K. C. Carroll. 2009. Pathogen profiling: rapid molecular characterization of Staphylococcus aureus by PCR/electrospray ionization-mass spectrometry and correlation with phenotype. J. Clin. Microbiol. 47: 31293137.
57. Zou, W.,, J. G. Frye,, C. W. Chang,, J. Liu,, C. E. Cerniglia, and, R. Nayak. 2009. Microarray analysis of antimicrobial resistance genes in Salmonella enterica from preharvest poultry environment. J. Appl. Microbiol. 107: 906914.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error