Ourania Raftopoulou

Listeria monocytogenes strains representing diverse serotypes, genotypes and sources were selected for sequence tagging. Strains were genetically tagged with unique genetic barcodes of approx. 30 nt cloned into the barcoding vector pTZ200.mix, which was then inserted in the same chromosomal locus in each strain. Stable chromosomal tagging was attained for 8 different strains representing serotypes 1/2a, 1/2b, and 4b. Sequencing of PCR products from the barcode- harboring fragments indicated unique sequence tags for each strain. The barcoded strains and their parental counterparts were assessed for growth, motility biofilm formation, hemolysis and virulence. Biofilm formation was assessed in polystyrene microwells with the crystal violet assay. To assess hemolytic activity, the strains were streaked on blood agar, incubated at 37^oC and visually examined over 36 h. Motility was appraised by spotting 10 ul of overnight cell suspensions on soft agar, incubating at 25^oC and visually comparing the spots over 36 h. Growth at 4, 15, 25 and 37^oC was examined by streaking the strains on BHI agar and comparing the size of single colonies after 15, 2, 2 and 1 days, respectively. For virulence assessments, larvae of Galleria mellonella were injected in the last left proleg with 10⁵ and 10⁶ CFU per larva, incubated at 37^oC and monitored for survival over 7 days. No significant (p<0.05) differences were noted in biofilm formation and virulence between barcoded and parental strains. However, noticeable differences were noted with certain strains in hemolytic activity. Specifically, 2010L-17234-4 (celery outbreak, serotype 1/2a) was consistently more hemolytic than its parental counterpart. Differences were also noted in motility between certain tagged and parental strains, with three of the tagged strains exhibiting either smaller or larger spots on soft agar than their parental counterparts. No significant differences were noted in colony size between the parental and tagged strains at 37^oC, while differences were noted for certain strains at the lower temperatures. The findings suggest that there should be some caution in using such barcoded derivatives, since certain phenotypic traits could have been impacted.