Removal of Listeria monocytogenes Biofilms with Bacteriophage P1003

Listeria monocytogenes is an important foodborne pathogen with a persistent ability to form biofilm matrices in the food processing environments. In this study, we have determined the ability of bacteriophage P100 to reduce L. monocytogenes cell populations under biofilm conditions by using 21 L. monocytogenes strains representing 13 different serotypes. There were considerable differences in the ability of various strains of L. monocytogenes to form biofilms, with strains of serotype 1/2a showing maximum biofilm formation. Irrespective of the serotype, growth conditions, or biofilm levels, the phage P100 treatment significantly reduced L. monocytogenes cell populations under biofilm conditions. On the stainless steel coupon surface, there was a 3.5to 5.4-log/cm reduction in L. monocytogenes cells by phage treatment. These findings illustrate that phage P100 is active against a wide range of L. monocytogenes strains in biofilm conditions. The persistence of Listeria monocytogenes biofilms on food and nonfood contact surfaces is the major attribute facilitating this pathogen’s environmental spread and subsequent contamination of ready-to-eat food products (11, 20). In a food processing plant, the common sites for L. monocytogenes isolations are floor drains, conveyor belts, stainless steel equipment surfaces, product transportation racks, and cold rooms (37). Prevailing conditions on these sites such as ample water content and food residues further provide a unique opportunity for pathogenic L. monocytogenes to form a biofilm matrix and reside therein (11). Though the adherence strength of different L. monocytogenes strains varies, the majority of L. monocytogenes strains of different serotypes are able to produce biofilm mass under defined laboratory conditions (11, 15). The genetic determinants required for L. monocytogenes biofilm formation are still relatively unknown (18), although screening of different insertion mutant libraries along with phenotypic characterization has produced some meaningful information. Some of the important genetic determinants involved in L. monocytogenes biofilm include surfaceadhering protein BapL (18), flagellar protein FlaA, and DNA-binding two-component response regulator DegU (22), SOS-controlled protein YneA (42), quorum-sensing– regulated peptidase AgrD (30), and the ability of some strains to produce truncated InlA over full-length InlA (12). L. monocytogenes cells present in a biofilm matrix show greater resistance to antimicrobial agents than those present in planktonic environments (26, 31). Examples of antimicrobial agents that have been tested for their efficacy against L. monocytogenes biofilms include alkaline and acidic electrolyzed water (3), octenidine hydrochloride (1), quaternary ammonium compounds, sodium hypochlorite, and hydrogen peroxide (26, 44). These findings report a 2to 4-log reduction in L. monocytogenes populations in biofilms and indicate that although sanitizer compounds aid in biofilm removal, the degree of biofilm removal depends on factors such as biofilm age, the surface on which biofilm is formed, and the substrate in which the biofilms were produced. In addition, repeated exposure to single disinfecting agents could also confer subsequent insensitivity to L. monocytogenes cells (26). Apart from chemically based treatments, Zhao et al. (46) used a competitive-exclusion strategy and demonstrated that the use of Lactococcus lactis and Enterococcus durans in poultry processing floor drains resulted in 2to 6-log reductions of L. monocytogenes compared with 8 log CFU/100 cm levels in control coupons containing 28-day-old biofilm cells. Another promising approach to control and eradicate biofilm formation is the use of bacteriophages as antibacterial agents (4, 33, 36, 46). Bacteriophages (phages) are viruses that infect bacterial cells specific for a target genus, serotype, or strain. Bacteriophages are ubiquitous in nature, and as many as 10 phage particles can be isolated from 1 g of soil or water (27). Phages have also been isolated from several food products such as meat, dairy, and vegetable products (2, 5, 16, 19, 43). All phages are obligate parasites, meaning that they rely on a specific host for propagation. For biocontrol strategies, lytic phages that have the ability to * Author for correspondence. Tel: 662-325-7697; Fax: 662-325-8728; E-mail: [email protected]. { Approved for publication as journal article no. J-11708 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University. Work was completed under project MIS-401100. 1519 Journal of Food Protection, Vol. 73, No. 8, 2010, Pages 1519–1524 Copyright G, International Association for Food Protection