Comparison of three enrichment schemes for the detection of low levels of desiccation-stressed Listeria spp. from select environmental surfaces

Abstract Three commonly used enrichment schemes were evaluated for the detection of Listeria spp. from environmental surfaces: one-step 48 h enrichment in buffered Listeria enrichment broth (BLEB); 26 h primary enrichment in University of Vermont modified (UVM) broth, followed by a transfer of 100 μl primary enrichment culture to Fraser broth (FB) for 24–48 h secondary enrichment; and 26 h primary enrichment in half-Fraser broth (HF), followed by a transfer of 100 μl primary enrichment culture to FB for 24–48 h secondary enrichment. Low levels (20–300 CFU/sample) of Listeria were artificially inoculated onto eight types of surfaces, desiccation stressed, and then collected by swab/sponge samplers for subsequent analysis. Results showed that low levels of desiccation-stressed Listeria recovered and grew rather slowly: 26 h primary enrichment in UVM and HF could not consistently yield 1 cell in 100 μl of enrichment culture, as demonstrated by enumeration of 100 μl primary enrichment culture and transfer of 100 μl enrichment culture to secondary enrichment. We further discovered that, using 20 samples per scheme and for certain Listeria strains on certain surfaces, one-step 48 h enrichment in BLEB generated significantly higher numbers of positive results than the two-step enrichment schemes when there were no competing background microflora. Higher numbers of positive results from two-step schemes were achieved by increasing the duration of primary enrichment for an additional 22 h. Therefore, to improve the detection of desiccation-stressed Listeria in the environment, we must allow sufficient time for primary enrichment and/or transfer a sufficient amount of primary enrichment culture to the secondary enrichment. The study also highlights the importance of using low levels of inoculum and physiologically injured cells for method comparison and evaluation.