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Ice Cream Method Validation, Detection of Listeria monocytogenes

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Over the last two years (2022-2023), eight recalls and two outbreaks (including one death) in were attributed to ice cream contaminated with Listeria monocytogenes in the US. While L. monocytogenes does not grow in a frozen state, these recent recall and outbreak events highlight the potential for this pathogen to survive and cause disease. Additionally, studies of frozen foods (ice cream, frozen vegetables) contaminated with L. monocytogenes have identified this organism can persist for up to a year with minimal reduction [1,2]. Given the rise in incidences of contamination, it is essential that your  L. monocytogenes rapid detection method has been assessed for ice cream analysis.

When selecting a method, it is important to consider if the existing validation data supports (i) analyzing ice cream at the desired test portion size and (ii) testing ice cream from a frozen vs. thawed state. Given the demand for shortened time-to-results, there is often a push to select a rapid method with the shortest incubation time. However, when testing ice cream, evaluating how the method performs with frozen products is important. Specifically, shortened incubation times (≤ 24 h), and testing pooled, larger test portions (>25 g) may reduce method sensitivity (i.e., lead to false negative results). Compared to refrigerated and ambient products, enriched ice cream samples take longer to reach incubation temperature; hence, a longer incubation time may be needed for growth to detectable levels. Additionally, increasing the portion size further increases the time needed to reach the incubation temperature. An ice cream study[3] evaluating L. monocytogenes detection from 125 g, 250 g, and 375 g test portion sizes using the ISO 11290-1 reference method yielded acceptable results for only the 125 g sample size. Importantly, the ISO method follows a two-step enrichment procedure (i.e., a primary followed by a secondary enrichment) with a total incubation time of 48 hours (h), which may not be prescribed in all available methods.

End users need to be aware that real-world method performance does not always mimic the performance seen in validation studies. For example, difficulty with cultural isolation of L. monocytogenes from a presumptive positive ice cream enrichment has been documented. A study[4] of naturally contaminated ice cream gave insight into why cultural isolation of L. monocytogenes from ice cream is not always successful (i.e., L. monocytogenes is not isolated following a rapid method presumptive positive). While all samples in the Ottesen et al. study[4] were naturally contaminated with L. monocytogenes, the background flora dominated the enrichment for the first 24 hours. Only after 24 h did L. monocytogenes grow to higher levels than the co-enriched background flora; at 48 h, L. monocytogenes was the dominant organism. Therefore, when a rapid detection method yields a presumptive positive, but the cultural confirmation does not yield an L. monocytogenes isolate, key factors to consider include: (i) at what time point was streaking performed (e.g., 24 h, 48 h), (ii) was a secondary enrichment step performed, and (ii) could L. monocytogenes growth be masked by the presence of background microflora. 

Interested in tracking food recalls? Learn more about our EMAP Consulting services, and contact us if you have any questions.  

References

[1]  Chen YI, Burall LS, Macarisin D, Pouillot R, Strain E, De Jesus AJ, Laasri A, Wang H, Ali L, Tatavarthy A, Zhang G. Prevalence and level of Listeria monocytogenes in ice cream linked to a listeriosis outbreak in the United States. Journal of Food Protection. 2016 Nov 1;79(11):1828-32.

[2]  Fay ML, Salazar JK, Stewart DS, Khouja BA, Zhou X, Datta AR. Survival of Listeria monocytogenes on Frozen Vegetables during Long-term Storage at-18 and-10° C. Journal of Food Protection. 2024 Jan 18:100224.

[3]  Jagadeesan B, Schmid VB, Kupski B, McMahon W, Klijn A. Detection of Listeria spp. and L. monocytogenes in pooled test portion samples of processed dairy products. International journal of food microbiology. 2019 Jan 16;289:30-9.

[4]  Ottesen A, Ramachandran P, Reed E, White JR, Hasan N, Subramanian P, Ryan G, Jarvis K, Grim C, Daquiqan N, Hanes D. Enrichment dynamics of Listeria monocytogenes and the associated microbiome from naturally contaminated ice cream linked to a listeriosis outbreak. BMC microbiology. 2016 Dec;16(1):1-1.

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