Isolation and detection of Listeria monocytogenes using fluorogenic and chromogenic substrates for phosphatidylinositol-specific phospholipase C

The BCM Listeria monocytogenes detection system (LMDS) consists of a select ive preenrichment broth (LMPEB), selective enrichment broth (LMSEB), select ively/differential plating medium (LMPM), and identification on a confirmat ory plating medium (LMCM). The efficacy of the BCM LMDS was determined usin g pure cultures and naturally and artificially contaminated environmental s ponges. The BCM LMPEB allowed the growth of Listeria and resuscitation of h eat-injured L. monocytogenes, The BCM LMSEB, which contains the fluorogenic substrate 4-methylumbelliferyl-myo-inositol-1-phosphate and detects phosph atidylinasitol phospholipase C (PI-PLC) activity, provided a presumptive po sitive test for the presence of pathogenic Listeria (L. monocytogenes and L . ivanovii) after 24 h at 35 degrees C. An initial inoculum of 10 to 100 CF U/ml of L. monocytogenes in BCM LMSEB yielded a fluorogenic response after 24 h. On BCM LMPM, L. monocytogenes and L. ivanovii were the two Listeria s pecies forming turquoise convex colonies (1.0 to 2.5 mm in diameter) from P I-PLC activity on the chromagenic substrate, 5-bromo-4-chloro-3-indoxyl-myo -inositol-1-phosphate. L. monocytogenes was distinguished from L. ivanovii by either its fluorescence on BCM LMCM or acid production from rhamnose. Fa lse-positive organisms (Bacillus cereus, Staphylococcus aureus, Bacillus th uringiensis, and yeasts) were eliminated by at least one of the media in th e BCM LMDS. Using a pure culture system, the BCM LMDS detected one to two L . monocytogenes cells from a sponge rehydrated in 10 ml of DE neutralizing broth. in an analysis of 162 environmental sponges from facilities inspecte d by the U.S. Department of Agriculture (USDA), the values for identificati on of L. monocytogenes by BCM LMDS and the USDA method were 30 and 14 sites , respectively, with sensitivity and specificity values of 85.7 and 100.0% versus 40.0 and 66.1%, respectively. No false-positive organisms were isola ted by BCM LMDS, whereas 26.5% of the sponges tested by the USDA method pro duced false-positive results.