Evaluation of the efficacy of antimicrobial agents to prevent the transfer of Listeria monocytogenes from existing biofilms to produce or processing surfaces

Listeria monocytogenes (Lm) is a foodborne pathogenic bacterium that can cause serious illness and even death in susceptible individuals. Outbreaks involving this pathogen have been associated with fruits, sprouts and vegetable row crops. Like most other bacteria, Lm can form biofilms or became part of biofilms with other bacteria on produce surfaces and surfaces in produce harvesting and processing environments. Once established in a biofilm, Lm has highly diminished susceptibility to antimicrobial agents and is difficult to eradicate. Cells surviving in such biofilms can detach and be carried to new surfaces where they can start the formation of a new biofilm or become part of an existing biofilm. It is therefore extremely important to prevent the transfer of cells from existing biofilms to previously uncontaminated surfaces on produce or processing equipment. The proposed study will examine the efficacy of antimicrobial agents to inactivate Lm released from existing biofilms and prevent the formation of new Lm-containing biofilms on produce and equipment surfaces.

Bacteria that are part of a biofilm community are considerably less susceptible to antimicrobial agents than their planktonic counterparts. It is therefore very difficult to eradicate biofilm-associated bacteria from surfaces of fruits and vegetables or harvesting and processing surfaces. Biofilm-associated bacteria surviving treatments with antimicrobials, such as hypochlorite, chlorine dioxide, hydrogen peroxide and non-oxidizing agents, are a source of bacteria that can be released from the biofilm and colonize other surfaces. The proposed research is focused on Listeria monocytogenes (Lm) and the efficacy of antimicrobial agents in preventing Lm released from biofilms to find refuge on produce and processing surfaces. Initially, Lm biofilms will be established on microtiter plate surfaces, and the effectiveness of antimicrobials in preventing transfer of the pathogen from the biofilms through media containing produce-derived organics will be determined. Antimicrobial efficacy in preventing transfer and survival of Lm originating from biofilms to surfaces already colonized by other bacteria will also be examined. Finally, transfer of Lm from biofilms established on produce and processing surfaces to previously Lm-free produce or processing surfaces will be determined in the presence and absence of antimicrobial concentrations previously determined to be effective in the microtiter plate assays. The studies will help produce producers and processors as well as regulatory agencies to determine if currently used antimicrobial agents are capable of preventing the transfer of Lm from existing sources to other surfaces and at which concentration prevention does occur.

1. In laboratory experiments, determine the concentrations of hypochlorite, hydrogen peroxide, peracetic acid and other permissible antimicrobials that are required to prevent the transfer of Lm from an existing biofilm to an uncontaminated surface when tap water or water with different organic loads is the transfer medium. 

2. Determine if Lm released from a biofilm is able to take refuge on a new surface coated with organic materials and bacteria. 

3. Validate the results by monitoring the transfer of Lm existing as biofilms on leafy greens and fruits and vegetables to uncontaminated produce or equipment surfaces in the presence of antimicrobials. Also, monitor the transfer from abiotic surfaces to leaves or produce surfaces.

The data collected in this study indicate that Listeria monocytogenes in biofilms cannot be reliably inactivated by antimicrobials at concentrations usually found in produce wash water. This observation was made when the wash medium was essentially free of organic compounds, except for those released by the biofilms. Therefore, it can be expected that the required concentrations in systems with organic loads will be even higher. Despite the observed survival of Lm in biofilms in the presence of relatively high concentrations of antimicrobials, Lm released from such biofilms did not show the same level of resistance as those associated with a surface. Lm appears to be released from biofilms in a state that makes them susceptible to inactivation by low concentrations of antimicrobials. The data stress the need to prevent the formation of biofilms as much as possible to prevent the creation of reservoirs for Lm in a facility; however, the data also indicate that as long as recommended concentrations of antimicrobials are maintained in wash water, it should be possible to prevent Lm from reaching existing biofilms or to transfer to produce being washed. Lm associated with soil appears to be difficult to inactivate, but further studies need to be done with different soils and soil levels to determine concentrations of antimicrobials that are effective against Lm associated with soil particles.