A streamlined procedure for advancing the detection and isolation of <i>Listeria monocytogenes</i> from artificially contaminated ground beef in a single working day.

Abstract

<i>Listeria monocytogenes</i>, a rod-shaped Gram-positive bacterium widely distributed in nature, can contaminate foods and represents a foodborne pathogen of public health significance causing a high mortality rate of 20%-30%. Rapid and reliable identification of foods and food-processing environments contaminated with <i>L. monocytogenes</i> is a crucial step in implementing effective intervention strategies to ensure food safety and limit the transmission of bacteria to humans. This study designed and refined a practical workflow to streamline and accelerate the detection of a low level of <i>L. monocytogenes</i> present in ground beef. The workflow coupled an abbreviated 5 h culture enrichment in PALCAM liquid medium with physical separation (filtration and centrifugation) to preprocess enrichment samples. Specific capture was achieved using magnetic separation with a bacteriophage endolysin-derived cell wall-binding domain in a Hyglos <i>Listeria</i> capture kit. Molecular detection was performed using a MicroSEQ <i>L. monocytogenes</i> RTi-PCR detection kit combined with a nested PCR strategy. Preprocessing of enrichment culture samples using a multi-stage filtration system constructed for the study or commercially available BagFilter Pull-up filter bags, in conjunction with centrifugation, enabled the recovery of ~30 colony-forming units (CFUs) from the enrichment culture of a 25 g ground beef sample artificially contaminated with 1 CFU of <i>L. monocytogenes</i>. Integration of magnetic separation into the workflow for capturing <i>L. monocytogenes</i> cells specifically from preprocessed samples and further cleaning up the samples yielded bacterial counts similar to those obtained by direct plating of preprocessed samples. The RTi-PCR-based molecular detection method integrated into the workflow was capable of detecting pure cultures of <i>L. monocytogenes</i> as low as 12.5 CFUs. Evaluation of the workflow using artificially ground beef demonstrated the consistent detection of <i>L. monocytogenes</i> within an 8 h workday in a 25 g sample unit containing the cell count as low as 2 CFU following a 5 h culture enrichment.<h4>Importance</h4>Consuming foods contaminated with the bacterial pathogen <i>Listeria monocytogenes</i> can lead to the development of human listeriosis, a severe and life-threatening foodborne illness. Timely detection of <i>L. monocytogenes</i> present at a low level in foods and food processing environments is a necessary measure to prevent the spread of the <i>Listeria</i>-associated illness. This study designed and evaluated a multi-step workflow for testing <i>L. monocytogenes</i> in artificially contaminated food samples. The workflow was composed of a short 5 h culture enrichment, filtration-based sample preprocessing, magnetic separation, a single-tube nested RTi-PCR, and culture plating. It allowed <i>L. monocytogenes</i> to be detected within 8 h from a 25 g ground beef sample containing the target cells as low as 2 colony-forming units, significantly improving and streamlining the detection methods for this important foodborne pathogen.