Identification of epidemiologic markers for Neisseria meningitidis using difference analysis.
The feasibility of identifying epidemiologic markers based solely on the identification of DNA fragments present in outbreak-associated isolates was investigated using Neisseria meningitidis (Nm) as a model system. The clonal structure of Nm has been well characterized using multilocus electrophoresis. In Canada, electrophoretic types ET1, ET5, ET9 and ET21 are being displaced from the natural population by type ET15, and the latter type is associated with an increased prevalence of serogroup C meningococcal disease. Difference analysis, which uses subtractive hybridization and polymerase chain reaction (PCR) amplification, was employed to identify amplifiable DNA fragments (amplicons) that differ between the ET15 and the ET1, ET5, ET9 and ET21 genomes. 14 amplicons were cloned which were further characterized by Southern blot analysis to identify six amplicons that represent fragments either unique to or highly polymorphic in the ET15 genome. Oligodeoxyribonucleotide primer pairs were designed for each of the six amplicons, and PCR amplification was used to determine their prevalence across a panel of 167 Nm isolates representative of other serogroups and ETs. Among group C isolates only two of the six amplicons, designated as A and G, were effective in discriminating ET15 from non-ET15 isolates. Amplicon A detects a deletion in the dhps gene which effectively differentiates sulfonamide-sensitive and -resistant serogroup C isolates. The frequency of amplicon A and G detection in the other serogroups and ETs was too great to facilitate their direct use as diagnostic markers for the differentiation of virulent Nm isolates.
National Laboratory for Bacteriology and Enteric Pathogens, Bureau of Microbiology, Ottawa, Ontario, Canada.
C A
CA
Strathdee
W M
WM
Johnson
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001483:Base Sequence; D002170:Canada; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics*; D004094:Dihydropteroate Synthase / Q000235:genetics; D004196:Disease Outbreaks; D005819:Genetic Markers; D006801:Humans; D008585:Meningitis, Meningococcal / Q000453:epidemiology*; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D005819:Genetic Markers; C083821:iroA protein, bacteria; D004094:Dihydropteroate Synthase