Quantitative Evaluation of Three Selective Enrichment Broths and Agars Used in Recovering Listeria Microorganisms.

ABSTRACT Three selective enrichment broths (FDA, University of Ver­mont and Dominguez Rodriguez) and lithium chloride-phenyle-thanol-moxolactam (LPM) agar, modified McBride (MMA) agar, Listeria selective agar (LSAM of Dominguez-Rodriguez), and Brain Heart Infusion (BHI) agar (as reference) were evaluated for their suitability to support the growth of six different species/ strains of Listeria (Listeria monocytogenes Scott A, L. monocytogenes V7, L. monocytogenes VPH-1, Listeria innocua, Listeria seeligeri, and Listeria ivanovii). All Listeria strains grew faster and yielded a higher number of cells in FDA enrichment broth. Based on MPN studies, 1 to 2.1 cells of L. monocytogenes and L. innocua were needed for visible colony formation, on all three selective and BHI agars after 48 h incubation of 37°C. The LPM agar was more inhibitory for L. seeligeri and L. ivanovii requiring 9.6 and 917 cells, respectively, as compared to 1 to 2.7 cells for the other agars. The effectiveness of a particular combi­nation among the selective enrichment broths and agars for recovering L. monocytogenes Scott A from inoculated cheese and meat samples was quantitated. Any enrichment broth combined with plating on LPM or LSAM agar gave 100% Listeria recovery as compared to 50 to 67% for plating on MMA agar. Both LPM and LSAM agars have also shown a superior performance to MMA agar in the recovery of naturally occurrin Listeriag from soft cheese and raw meat. The use of a secondary broth enrichment step improved the recovery of Listeria spp. from meat samples. Listeria monocytogenes has been involved in recent food-associated outbreaks of listeriosis (20) and has been isolated from a variety of foods (20), which may serve as vehicles of transmission of listeriosis to humans. L. mon­ocytogenes is widely distributed in the environment and foods and its ability to grow at refrigeration temperatures has increased our interest for the detection of contaminated foods. L. monocytogenes is usually present in foods at low concentrations being generally overgrown by the background microflora. This has made attempts to isolate the pathogen by direct plating unsuccessful in most cases. Since cold en­richment (2) at 4°C and subculturing weekly for up to six 'Current address: Departmento de Patologia Animal I (Sanidad Animal), U.D. Microbiologia lnmunologw, e Facultadde Veterinaria, Universidad Complutense, 28040 Madrid, Spain. months is unsuitable for routine examination of foods produced commercially, more rapid procedures for the isolation of L. monocytogenes have (2,5). been developedBasically, most of these methods include an enrichment step in appropriate broths with selected microbial inhib­itors, to obtain higher populations of Listeria cells. The cells are then detected by gene probes and ELISA tech­niques or plating on selective agars (9,14,15). Direct dem­onstration of the pathogen in food by immunofluorescence has also been reported (21). Numerous selective enrichment broths, agars, and pro­cedures for Listeria isolation from contaminated samples have been proposed and reviewed recently (2,19). Of these procedures and media, two have had a wide acceptance for routine detection of Listeria in foods and food processing environments, namely the U.S. Food and Drug Administra­tion (FDA) (18) and U.S. Department of Agriculture (USDA) (16) methods. A limited number of papers have been pub­lished which compare the various procedures and media for Listeria recovery from foods (1,7,12,13,14). In all of these studies conclusions were based exclusively on the degree of Listeria recovery from inoculated or market foods, while data on the actual behavior of Listeria spp. in the enrich­ment broths and selective agars, with respect to the number of cells needed for growth and rates of growth, are lacking. The purpose of this work was mainly to generate data in this particular area with emphasis on the behavior of three strains of Listeria monocytogenes and one strain each of Listeria innocua, Listeria ivanovii, and Listeria seeligeri in the FDA and USDA selective enrichment broths and agars. In addition, we included an enrichment broth pro­posed by Dominguez Rodriguez et al. (4) for recovery of Listeria from heavily contaminated samples and a selective agar which has shown a high efficacy for Listeria isolation (3,5).