Assessment of Reference Method Selective Broth and Plating Media with 19 Listeria Species Highlights the Importance of Including Diverse Species in Listeria Method Evaluations

Reference methods developed for detection of Listeria monocytogenes are commonly used for detection of Listeria at the genus level. Improved method performance data are needed because this genus has expanded from 6 to 26 species and now includes several Listeria sensu lato species, which can have phenotypes distinct from those of Listeria sensu stricto. We evaluated growth of 19 Listeria species, including 12 recently described Listeria sensu lato species, using the media specified by (i) the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual, (ii) the U.S. Department of Agriculture (USDA) Microbiology Laboratory Guidebook, and (iii) the International Organization for Standardization (ISO). The FDA broth enrichment procedure allowed all species to grow to detectable levels (≥4 log CFU/mL), yielded the highest mean growth (7.58 log CFU/mL), and was the only procedure with which no Listeria sensu lato species yielded significantly higher growth than did a comparison Listeria sensu stricto species. With the USDA and ISO broth enrichment procedures, several Listeria sensu lato species yielded significantly higher growth than did either Listeria seeligeri or Listeria ivanovii, suggesting that these two Listeria sensu stricto species could be outgrown by Listeria sensu lato species. On selective and differential agar media, L. seeligeri, L. ivanovii, and Listeria grayi produced colonies with atypical morphology and/or growth of these species was inhibited (which may lead to incorrect classification of a sample as negative), whereas several newly described Listeria sensu lato species grew to high levels and produced colonies with typical morphology. Overall, our study results indicate that the ability to detect various Listeria species can be impacted by the specific broth and selective and differential agar used. Our data can help guide selection of appropriate media and detection methods for environmental Listeria monitoring programs and methods that are most likely to detect the targeted Listeria groups (e.g., Listeria sensu stricto, which appear to be the most appropriate index organisms for the pathogen L. monocytogenes).