Validation of a Novel Secondary Enrichment Broth for Resuscitating Viable but Nonculturable (VBNC) Salmonella spp. in Environmental Samples

Introduction: The survival capacity of Salmonella increases the probability of the contamination of agricultural commodities and their processing environments. Sampling food production and processing environments can be difficult, where bacteria may enter a non-viable state in order to protect themselves from environmental stress. Traditional culture methods are effective for food testing, but modifications need to take place in order to allow a more efficient recovery of stressed cells from the environment. Studies have shown that Salmonella is able to enter a VBNC state, regain culturability and cause disease once it has entered the intestinal tract of a host. 

Purpose: The objective of this study was to evaluate the effectiveness of a modification of Rappaport Vassiliadis broth and compare it to the recommended secondary enrichment from regulatory agencies for the recovery of VBNC Salmonella cells in environmental samples.

Methods: Samples obtained from a sweet potato field fertilized with composted chicken litter (soil, n=28), and a catfish farming area (pond water and mud, n=40) were pre-enriched in Buffered Peptone Water, transferred to Rappaport Vassiliadis, Tetrathionate, and biochemically modified Rappaport Vassiliadis, incubated at 41.5°C and further analyzed using USDA/FSIS and BAX real-time PCR methods. Presumptive colonies separated on Brilliant Green Agar and Xylose Lysine Deoxycholate agar were confirmed by PCR. 

Results: Detection rates for the soil samples were 43% on real time PCR, 54% on Rappaport Vassiliadis, 43% on Tetrathionate, and 61% on modified Rappaport Vassiliadis, and for pond water and mud these were 13% on real time PCR, 8% on Rappaport Vassiliadis, 8% on Tetrathionate, and 23% on modified Rappaport Vassiliadis.  

Significance: Modified Rappaport Vassiliadis could be a good alternative enrichment broth to resuscitate VBNC cells that would go undetected with traditional methods and that could regain viability, contaminate foods, and cause foodborne outbreaks.