Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 12, 2002
Publication Date: February 20, 2003
Citation: PALUMBO, J.D., BORUCKI, M.K., MANDRELL, R.E., GORSKI, L.A. 2003 SEROTYPING OF LISTERIA MONOCYTOGENES BY ENZYME-LINKED IMMUNOSORBENT ASSAY AND IDENTIFICATION OF MIXED-SEROTYPE CULTURES BY COLONY IMMUNOBLOTTING. JOURNAL OF CLINICAL MICROBIOLOGY. v. 41 p 564-571 Interpretive Summary: Listeria monocytogenes is an important foodborne pathogen of humans. In order to understand the movement of the bacterium from food to humans during the course of a disease outbreak, methods for identifying subgroups of Listeria have been developed. One such method, serotyping, separates Listeria monocytogenes into 12 different subgroups by each subgroup's pattern of reaction to a set of antibodies produced for that purpose. This method has been in use for decades, but in its current form, the bacterium-antibody reactions are judged visually, which may be a source of inaccuracy in the method. In this paper, we describe a new serotyping method, using the same antibodies as the current method. In the new method, positive bacterium-antibody reactions are detected by a reaction that gives a yellow product. The intensity of the yellow product can be measured to give an indirect measurement of the strength of the bacterium-antibody reaction. Using this method, we positively identified all 12 possible subgroups of Listeria monocytogenes in a culture collection of 201 different strains. Comparing the previous subgrouping method to the new subgrouping method, we found that the two methods assigned the same subgrouping to 89 of 101 bacterial strains tested. Using the same antibodies, we also developed a method to determine whether a culture of Listeria monocytogenes contains more than one subgroup type. These methods will benefit other laboratories performing subgrouping analyses of Listeria monocytogenes, because they reduce the variability of the previous method and the amount of antibodies used per bacterial strain, resulting in a significant cost savings. Also, these methods will allow researchers to determine whether there may be more than one subgroup present in a food contaminated with Listeria monocytogenes.
Technical Abstract: Routine analysis of Listeria monocytogenes by serotyping using traditional agglutination methods is limited in use because of the expense and limited availability of commercially prepared antisera and intra- and interlaboratory discrepancies arising from differences in antiserum preparation and visual determination of agglutination. We have adapted a commercially available set of L. monocytogenes antisera to an ELISA format for high-throughput, low-cost serotype determination. Rather than subjective visualization of agglutination, positive antigen/antiserum reactions were scored by a quantitative, colorimetric reaction. ELISA serotyping of 89 of 101 L. monocytogenes isolates agreed with slide agglutination serotyping data, and 100 previously uncharacterized isolates were serotyped unambiguously using the ELISA method. In addition, mixed-serotype cultures of L. monocytogenes were identified by a colony immunoblot procedure, in which serogroup 1/2 and serogroup 4 colonies were discriminated by differential staining.