High-Resolution Genotyping of Campylobacter Species by Use of PCR and High-Throughput Mass Spectrometry

Abstract

In this work we report on a high-throughput mass spectrometry-based technique for the rapid highresolution identification of Campylobacter jejuni strain types. This method readily distinguishes C. jejuni from C. coli, has a resolving power comparable to that of multilocus sequence typing (MLST), is applicable to mixtures, and is highly automated. The strain typing approach is based on high-performance mass spectrometry, which "weighs" PCR amplicons with enough mass accuracy to unambiguously determine the base composition of each amplicon (i.e., the numbers of A's, G's, C's, and T's). Amplicons are derived from PCR primers which amplify short (<140-bp) regions of the housekeeping genes used by conventional MLST strategies. The results obtained with a challenge panel that comprised 25 strain types of C. jejuni and 25 strain types of C. coli are presented. These samples were parsed and resolved with demonstrated sensitivity down to 10 genomes/PCR from pure isolates.Food-borne pathogens are estimated to cause 76 million illnesses annually in the United States alone (19), and considering the impact on society from health care costs and lost productivity, the time and effort expended to understand the epidemiology of the underlying agents are well justified. Campylobacter has been identified as one of the leading causative bacterial agents associated with food-borne illnesses in the United States as well as worldwide (1,3,9,10,18,21,29). It is believed that many of these cases of illness are due to improperly handled poultry products, as Campylobacter contamination rates have been reported to be as high as 71% in commercial chicken products (31). The underlying etiology of 90% of the cases of campylobacteriosis is associated with Campylobacter jejuni, and the majority of the remaining cases are related to Campylobacter coli (11). Although they are not generally considered severe for healthy individuals, septicemia, meningitis, abortions, Guillian-Barré syndrome, and deaths have occurred (2, 23).The routine identification of infections or contamination typically relies on culture-based protocols (16). Culturing of these gram-negative, microaerophilic organisms is difficult and often requires several days to weeks; moreover, biochemical strain typing is not always accurate (24, 25). Alternative testing methods can include matrix-assisted laser desorption ionization-time of flight mass spectrometry biomarker analysis (8, 17), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, phage typing, resistotyping, serotyping, and PCR and genomic analysis (21).Campylobacter jejuni and C. coli PCR-multilocus sequence typing (MLST) has been at the forefront of recent research in this arena and is based on the characterization of genomic heterogeneity in highly polymorphic regions of seven housekeeping genes (4,20,22). MLST applies a phylogenetic ordering of strains that is nearly unambiguous as a means of identification, and a detailed database is being assembled to define strain sequence...

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