AEM Accepts, published online ahead of print on 22 June 2007

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Appl. Environ. Microbiol. doi:10.1128/AEM.00114-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A Microplate Fluorescence Assay to Measure Adherence to Abiotic Surfaces in Strains of Listeria monocytogenes from Meat and Meat Processing Plants

Rachel Gamble and Peter M. Muriana^*

Department of Animal Science, and The Oklahoma Food & Agricultural Products Research & Technology Center, Oklahoma State University, Stillwater, OK 74078-6055

^* To whom correspondence should be addressed. Email: peter.muriana{at}okstate.edu.

Listeria monocytogenes is a significant foodborne pathogen that is capable of adhering and producing biofilms on processing equipment making it difficult to eliminate from meat processing environments and allowing for potential contamination of ready-to-eat (RTE) products. We devised a fluorescence-based microplate method for screening isolates of L. monocytogenes for their ability to adhere to abiotic surfaces. Strains of L. monocytogenes were incubated for 2 days at 30^oC in 96-well microplates and washed in a plate washer. Retained cells were incubated for 15 min at 25^oC with 5,6-carboxyfluorescein diacetate (5,6-CFDA), washed again, and then fluorescence was read with a plate reader. Several enzymatic treatments (protease, lipase, and cellulase) were effective in releasing adherent cells from microplates and this process was used for quantitation on microbiological media. Strongly adherent strains of L. monocytogenes were identified that had 15,000-fold higher levels of fluorescence and 100,000-fold higher plate counts in attachment assays than weakly adherent strains. Strongly adherent strains of L. monocytogenes adhered equally well to 4 different substrates (glass, plastic, rubber, stainless steel), showed high level attachment on microplates at 10^o, 20^o, 30^o, and 40^oC, and showed significant differences with weakly adherent strains when examined by scanning electron microscopy (SEM). A greater incidence of strong adherence was observed for strains isolated from RTE meats than those isolated from environmental surfaces. Analysis of surface adherence among Listeria isolated from processing environments may provide a better understanding of molecular mechanisms involved in attachment and solutions to eliminate them from food processing environments.