Evaluation of Listeria Special Broth II (LSB II) for Recovery and Detection of Non-Stressed and Heat Stressed Listeria spp.

Gabriel Sanglay, Ryan Hartpence, Rania Chitour, Bryan Roach, and Daniel Smieszek.

Nestle Quality Assurance Center, Dublin, OH

Introduction: LSB II is Bio-Rad Laboratories’ latest second-generation enrichment media that allows for detection of Listeria spp at 37°C at 18-24 hours post-incubation on the iQ-Check PCR platform. NQAC Dublin performed initial evaluations on this medium and is now pursuing full implementation of this method.

Purpose: The objectives of this study were to:

1) Assess the performance of LSB II for detection of healthy and heat-stressed L. monocytogenes and L. innocua at minimum and maximum incubation times (18 and 24 hours) in validated and non-validated enrichment volumes (225ml and 1125ml, respectively).

2) Perform implementation and (food) item verification via the estimated limit of detection at 50% (eLOD50) of eight different ISO food categories per ISO 16140-3:2021 to claim a broad scope of foods.

Methods:

1) Healthy and heat-stressed cultures (treated at 56°C for 15 minutes) were inoculated into 225 or 1125ml of prewarmed LSB II and incubated at 37°C for 18 and 24 hours. At both time points, aliquots were taken for enumeration onto RAPID’L.mono agar, and for PCR analysis on the iQ-Check Listeria spp. and L. monocytogenes II kits. Experiments were replicated three times on three different days, and statistical analyses were performed using Minitab’s General Linear Model function.

2) For implementation and (food) item verification, the eLOD50 was conducted using protocol 1 in ISO 16140-3:2021. Healthy cultures of L. monocytogenes (5 strains), L. innocua, L. welshimeri, and L. ivanovii subsp. londoniensis were used for these studies.

Results:

1) Both healthy cultures of L. monocytogenes and L. innocua grew to 6.81 and 6.89 log CFU/ml after 18hrs incubation, respectively, and greater than 9 log after 24hrs incubation in 225ml enrichments. In the 1125ml enrichment volume, concentrations of healthy cultures were around ≥ 1 log lower than the 225ml volume. Heat-stressed L. monocytogenes and L. innocua grew to around 3-4 log CFU/ml in both enrichment volumes after 18hrs incubation, and 6-7 logs after 24hrs. Over 3 replicates, L. monocytogenes and L. innocua were successfully detected as soon as 18 hrs of incubation in the smaller enrichment volume, however the larger enrichment volume required longer incubation times for successful detection. The effects of culture type, stress, enrichment volume, and incubation time were statistically significant (p < 0.05).

2) The observed eLOD50 for all eight food categories ranged from <0 to <1 and were determined to be acceptable for LSB II and the iQCheck Listeria spp/L. monocytogenes PCR platform.

Significance: LSB II demonstrated great potential for rapid detection of Listeria for routine testing. NQAC Dublin will also be performing larger test portion (125g) validation in accordance with the draft guidelines set forth in ISO 16140-4:20/FDAM 1.

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