Listeria monocytogenes lineage III represents a diverse subset that is generally associated with non-human mammalian infection, although some isolates are capable of infecting humans. A cluster of virulence genes (prfA, plcA, hly, mpl, actA, and plcB), known as the prfA cluster, was sequenced in eleven lineage III isolates from food, animal, and human clinical cases, and analyzed in conjunction with forty previously sequenced isolates from lineages I (n = 18), II (n = 21), and III (n = 1). The lineage III isolates represent three previously identified lineage III subsets known as lineage IIIA, IIIB, and IIIC.

Evolutionary analysis identified intragenic positive selection in actA and plcA, and intergenic positive selection in the non-coding regions flanking hly. Positive selection in actA occurred in many positions previously recognized for their interactions with proteins involved in actin tail polymerization. Also, one positively selected site upstream of hly was located in its -35 promoter region. Additionally, horizontal gene transfer was recognized in actA, plcB, and mpl. The majority of events involved actA fragments transferred between lineages IIIA and II. Lineage IIIB did not partipate in any inter-lineage recombination events, but may have donated an mpl fragment to lineage IIIC. Neighbor joining phylogenies suggest that lineage III subsets IIIA and IIIC are monophyletic with lineage I, but that lineage IIIB is not. Therefore, due to its lack of horizontal gene transfer, and distant phylogenetic relationship to the rest of L. monocytogenes, lineage IIIB may represent a distinct subspecies.