Comparison of three neutralizing broths for environmental sampling of low levels of Listeria monocytogenes desiccated on stainless steel surfaces and exposed to quaternary ammonium compounds

Abstract

Background: An effective environmental sampling method involves the use of a transport/neutralizing broth with the ability to neutralize sanitizer residues that are collected during sampling and to maintain viability of stressed Listeria monocytogenes (Lm) cells.

Results: We applied Lm onto stainless steel surfaces and then subjected Lm to desiccation stress for 16-18 h at room temperature (RT, 21-24 °C). This was followed by the subsequent application of Whisper™ V, a quaternary ammonium compound (QAC)-based sanitizer, diluted to 400 ppm and 8000 ppm of active quat, for 6 h. We then sampled Lm with sponges pre-moistened in three transport broths, Dey/Engley (D/E) broth, Letheen broth and HiCap™ broth, to generate environmental samples that contained sanitizer residues and low levels of stressed Lm, which were subsequently analyzed by an enrichment-based method. This scheme conformed with validation guidelines of AOAC International by using 20 environmental test portions per broth that contained low levels of Lm such that not all test portions were positive (i.e., fractional positive). We showed that D/E broth, Letheen broth and HiCap™ broth performed similarly when no quat or 400 ppm of quat was applied to the Lm contaminating stainless steel surfaces. However, when 8000 ppm of quat was applied, Letheen broth did not effectively neutralize the QAC in the samples. These comparisons were performed on samples stored under three conditions after collection to replicate scenarios of sample transport, RT for 2 h, 4 °C for 24 h and 4 °C for 72 h. Comparisons under the three different scenarios generally reached the same conclusions. In addition, we further demonstrated that storing Letheen and HiCap™ broths at RT for two months before sampling did not reduce their capacity to neutralize sanitizers.

Conclusions: We developed a scheme to evaluate the ability of transport broths to neutralize QAC sanitizers. The three transport broths performed similarly with a commonly used concentration of quat, but Letheen broth could not effectively neutralize a very high concentration of QAC. The performance of transport broths was not significantly affected under the assessed pre-sampling and post-sampling storage conditions.

Keywords: Desiccation; Listeria monocytogenes; Sanitizer; Stainless steel surface; Transport broth.

Fig. 1

Three sets of experiments were performed in this study. The first set compared the three neutralizing broths without using Whisper™ V, a quaternary ammonium compound (QAC)-based sanitizer. The second set compared the three neutralizing broths by exposing inoculated Lm to QAC. Two quat concentrations were used, 400 ppm and 8000 ppm to represent a common concentration and a very high concentration. A pilot experiment was performed before this one to determine the appropriate combinations of Lm inoculum level and quat concentration that could yield fractional positive results in an AOAC validation scheme. After sponge sampling, three storage conditions were used to mimic same day sampling and enrichment-based analysis (i.e., 2 h at RT), enrichment-based analysis after overnight delivery of environmental samples (24 h at 4 °C) and enrichment-based analysis after over-the-weekend delivery of environmental samples (72 h at 4 °C). For the first two sets of experiments, three transport broths were stored at 4 °C before the experiments. The third set of experiments evaluated the stability of Letheen broth and HiCap broth stored at RT before sampling. Two separate evaluations were used because the inoculation levels and quat concentrations suitable to evaluate the two broths were different. The first one compared Letheen broth stored at RT and at 4 °C with D/E broth stored at 4 °C, and the second one compared HiCap broth stored at RT and at 4 °C with D/E broth stored at 4 °C. For the first and third set of experiments, we did not perform the analysis simulating the scenario of storing the sponge samples at 4 °C for 24 h due to logistical reasons