Strain Typing and Characterization Strain typing is a critical tool for determining the relatedness of individual pathogenic isolates, allowing epidemiologists to monitor the spread of disease and to detect changes in virulence or host range. Traditional strain typing and characterization techniques are based on methodologies such as phage typing or serotyping that often are unable to distinguish between genetically similar strains, especially at the level of small nucleotide differences. Genotypic approaches, including ribotyping and PFGE (Pulsed Field Gel Electrophoresis), provide greater discrimination in most cases, but are also labor-intensive and time-consuming. A new approach to characterizing genetic differences in previously indistinguishable isolates of the same or related species employs GeneChip microarrays to
classify strains by antibiotic resistance, virulence factors, or single nucleotide polymorphisms.
Determination of antibiotic resistance or sensitivity is of critical importance in bacterial infections, while characterization of new antibiotic resistance mutations allows elucidation of resistance mechanisms. Sougakoff et al. used a CustomSeq Array to characterize 41 rifampicin-sensitive and 59 rifampicin-resistance strains of Mycobacterium tuberculosis. Eleven distinct amino acid changes were identified in the rpoB (β-subunit of RNA polymerase) locus among the 59 resistant strains, as well as a single residue deletion in one of the sensitive strains. The resequencing array results were confirmed by conventional sequencing methods with 100% concordance. This study demonstrates the ability of the array to detect not only the presence of mutations in rpoB that confer rifampicin-resistance, but also to define the nucleotide changes responsible for the resistant phenotype, in a rapid (8 hour) protocol.
Recently, Dunman et al. compared the ability of the GeneChip S. aureus Array to accurately discriminate 21 oxacillin-resistant isolates of Staphylococcus aureus, representing strains of eight U.S. oxacillin- and methicillin-resistant lineages, to that of ribotyping and PFGE, both standard molecular epidemiological methods. Although strain clustering of the 21 isolates was similar among the three methods, the S. aureus Array results provided a higher level of discrimination of strains within the same cluster, especially on such genetic elements as virulence factors, antimicrobial resistance determinants, and agr (accessory gene regulator) type. This provides a highly discriminative genotyping procedure and simultaneously provides the ability to identify specific loci that are present or absent within a given S. aureus strain.
In another study, scientists at the United Kingdom’s Health Protection Agency (HPA) in Manchester used a CustomSeq Array containing genomic sequences that vary between different meningitis subtypes to classify different isolates of Neisseria meningitidis. Using the resequencing array, Corless et al. were able to correctly classify 45 samples that were previously identified by traditional methods, but more importantly, they were able to classify 12 previously unclassifiable samples into existing meningitis serotypes. Traditionally, the HPA has classified meningitis using immunoassays to identify serotypes in combination with DNA sequencing to identify sub-serotypes. In addition to being more accurate than the traditional serotyping methods, resequencing microarrays provide results in just 48 hours, much faster than their current methods. This resequencing array can now be used not only to rapidly type new meningitis isolates, but for epidemiological studies and vaccine research as well.
GeneChip arrays have also been applied to strain typing of biothreat agents to generate data which could be used to determine the source of the strain for forensic attribution purposes in criminal investigations. In a recent study, Zwick et al. used a CustomSeq Array to analyze the genomic sequence of a panel of 56 Bacillus anthracis strains - more than 3.1 megabases in total - and identified 37 novel SNPs. They also found that sequence data generated from the microarray had a very low error rate, in terms of replication in base calling ability, equivalent to a Phred 60 score, much higher than that typically obtained with conventional sequencing. At the same time, the error rates were less than 10-7, much lower than data from conventional sequencing methods, but the project took the group only 12 weeks to complete. Using traditional methods, sequencing this many strains would have taken 10 times as long and required 10 times the resources, demonstrating that the resequencing arrays represent a
rapid and cost-effective means of generating high-quality sequence data for strain typing purposes.
References
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Sougakoff, W. et al. Use of a high-density DNA probe array for detecting mutations involved in rifampicin resistance in Mycobacterium tuberculosis. Clin. Microbiol. Infect.10: 289-294 (2004).
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Dunman, P.M. et al. Uses of Staphylococcus aureus GeneChips in genotyping and genetic composition analysis. J. Clin. Microbiol.42: 4275-4283 (2004).
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Corless, C. et.al., Meningococcal Reference Unit, Health Protection Agency North West, Manchester Royal Infirmary, Oxford Road, Manchester, UK (2006). Submitted.
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Zwick, M.E. et al. Microarray-based resequencing of multiple Bacillus anthracis isolates. Genome Biol.6: R10 (2004).
Related Scientific Publications
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Couzinet, S. et al. Evaluation of a high-density oligonucleotide array for characterization of grlA, grlB, gyrA and gyrB mutations in fluoroquinolone resistant Staphylococcus aureus isolates. J. Microbiol. Methods60: 275? 279 (2005).
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van Leeuwen, W.B. et al. Multilocus sequence typing of Staphylococcus aureus with DNA array technology. J. Clin. Microbiol.41: 3323-3326 (2003).
Strain typing is a critical tool for determining the relatedness of individual pathogenic isolates, allowing epidemiologists to monitor the spread of disease and to detect changes in virulence or host range. Traditional strain typing and characterization techniques are based on methodologies such as phage typing or serotyping that often are unable to distinguish between genetically similar strains, especially at the level of small nucleotide differences. Genotypic approaches, including ribotyping and PFGE (Pulsed Field Gel Electrophoresis), provide greater discrimination in most cases, but are also labor-intensive and time-consuming. A new approach to characterizing genetic differences in previously indistinguishable isolates of the same or related species employs GeneChip microarrays to
classify strains by antibiotic resistance, virulence factors, or single nucleotide polymorphisms.
GeneChip arrays have also been applied to strain typing of biothreat agents to generate data which could be used to determine the source of the strain for forensic attribution purposes in criminal investigations. In a recent study, Zwick et al. used a 
