Influence and characterization of microbial contaminants associated with the FDA BAM method used to detect Listeria monocytogenes from romaine lettuce.

摘要

Over the past few decades in the US, fresh produce commodities have become increasingly prevalent vehicles for the attribution of foodborne illness. Recent outbreaks of the bacterial foodborne pathogen Listeria monocytogenes linked to fresh produce highlight this immediate issue facing food safety. The most widely used method to screen L. monocytogenes from food matrices in the US is the Bacteriological Analytical Manual (BAM) developed by the Food and Drug Administration (FDA). Detection of this pathogen from all foods is primarily accomplished by using four FDA approved Listeria selective media: Oxford (OXA), modified Oxford (MOX), Lithium chloride-phenylethanol-moxalactam fortified with esculin and iron (LPM), or PALCAM. Currently, there is a scarcity of evaluations concerning methods for isolation of L. monocytogenes from produce items. Thus, the first objective of this thesis work was to assess traditional FDA media and the commercial medium RAPID'L.mono for their use in detecting L. monocytogenes from the popular fresh produce item Romaine lettuce. Our results revealed that all four FDA media readily select for bacteria that based on their growth on the selective media appear to be L. monocytogenes but in fact belonged to other genera. The presence of these false positives ultimately limited the utility of each medium to detect Romaine lettuce samples that were found to be negative for L. monocytogenes. The commercial medium RAPID'L.mono was very accurate for detecting L. monocytogenes, as no false positives were characteristic of the pathogen on this medium. Testing false positives across media revealed that isolates recovered from MOX, OXA and PALCAM displayed broad positive behavior on other media. In contrast, the majority of isolates collected from LPM were found to have positive behavior restricted to that medium alone. The second objective of this thesis work was to perform whole genome sequencing of false positives taxonomically identified as Cellulomonas spp. to recover phylogenetic insights, determine how isolates survive selective plating and identify putative antibiotic target genes. Our phylogenetic analysis strongly supported that our isolates are species within the genus Cellulomonas. Resistance or susceptibility to antibiotics utilized in FDA media may be conferred by gene repertoires unique to certain isolates. We identified one potential antibiotic target gene present in Cellulomonas isolates that can be considered for future development of a selective medium to eliminate these false positives.