Purpose: To evaluate the impact of stress-mediated filamentation on the utility of various cultural and molecular techniques for detection and characterization of Salmonella.
Methods: Five Salmonella strains, including two peanut butter outbreak-associated isolates and an E. coli control were grown under four filament-inducing conditions of salt- or cold-stress in liquid or solid media. Additional non-filamented Salmonella controls were also prepared. These cultures were characterized by 1) Comparative quantification by optical density, plating and qPCR, 2) Combined nucleic acid staining and Flow Cytometry (FCM), followed by qPCR of sorted populations, 3) Peptide nucleic acid-based fluorescence in situ hybridization (PNA-FISH) and 4) Reverse transcriptase-qPCR (RT-qPCR) for measuring flagellar gene expression.
Results: Comparison of plating and qPCR results revealed a disconnect between cultural and molecular methods, with qPCR yielding higher apparent quantification than plating for filamented Salmonella. Recovery of filaments on XLT-4 was lower than on TSA, suggesting lowered fitness or injury. Stress-filamented treatments grouped into four distinct subpopulations according to nucleic acid content, and qPCR on FCM-sorted subpopulations highlighted the impact of cellular heterogeneity on molecular testing results. Finally, filaments contained sufficient rRNA to allow detection by Salmonella-specific PNA-FISH, but transcription of flagellar genes (fliC, fljB) was dramatically decreased compared to controls.
Significance: Our results highlight both the utility and drawbacks of existing cultural and molecular methods for detection of stress-filamented salmonellae. We expect our work will inform the use of appropriate tools for detecting the presence of such filaments in foods.