Multilocus fingerprinting of Listeria monocytogenes by sequence specific labeling of DNA probes combined with array hybridization

Summary

Multi locus sequence typing (MLST) is emerging as an alternative typing technique. MLST is based on sequence determination of several different genetic loci and the application of the variable sites in the DNA sequences to determine the relatedness between the different strains analyzed. We have developed an alternative MLST approach that targets the variable genetic changes directly in a DNA array format. Our approach is based on DNA array hybridization in combination with sequence specific labeling of oligonucleotide probes. Listeria monocytogenes was chosen for the development of the assay. This organism represents a major challenge in modern food production. The three virulence genes hlyA, iap and flaA were targeted. Five probes were constructed for the hlyA gene, 8 for the iap gene and 4 for the flaA gene. Reproducible signal profiles were obtained for 14 selected L. monocytogenes strains. The profiles were used in a maximum parsimony phylogenetic reconstruction. This analysis revealed that the strains could be divided in 7 different profiles, consisting of two statistically supported main clusters. One of these clusters could be divided into two subgroups. Furthermore, comparisons with strain serotypes and Amplified Fragment Length Polymorphism (AFLP) data gave good correlations. In conclusion, our DNA array-based MLST method is both a promising tool to fingerprint L. monocytogenes, and bacteria in general.