Isolation rates for Listeria monocytogenes and the other Listeria spp. typically improve when samples are enriched in more than one primary enrichment medium. This study evaluated the abilities of two primary enrichment media, University of Vermont-modified Listeria enrichment broth (UVM) and Listeria repair broth (LRB), to recover different ribotypes of Listeria spp. from raw meat and poultry samples. Forty-five paired 25-g retail samples of ground beef, pork sausage, ground turkey, and chicken (160 samples) underwent primary enrichment in UVM and LRB (30؇C for 24 h) followed by secondary enrichment in Fraser broth (35؇C for 24 and 40 h) and plating on modified Oxford agar. After 24 h of incubation at 35؇C, 608 Listeria colonies from selected positive samples were biochemically confirmed as L. monocytogenes (245 isolates), L. innocua (276 isolates), and L. welshimeri (89 isolates) and then ribotyped with the automated Riboprinter microbial characterization system (E. I. du Pont de Nemours & Co., Inc.). Thirty-six different Listeria strains comprising 16 L. monocytogenes (including four known clinical ribotypes), 12 L. innocua, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type; two UVM and two LRB isolates per sample). Twenty-six of 36 (13 L. monocytogenes) ribotypes were detected with both UVM and LRB, whereas 3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were observed with only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocytogenes), 21 (12 L. monocytogenes), 20 (9 L. monocytogenes), and 19 (11 L. monocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes confined to a particular product. More importantly, major differences in both the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third set of six samples per product type was examined from which two Listeria isolates were obtained by using only one of the two primary enrichment media, UVM and LRB failed to detect L. monocytogenes (both clinical and nonclinical ribotypes) in two and four samples, respectively. These findings stress the importance of using more than one primary enrichment medium and picking a sufficient number of colonies per sample when attempting to isolate specific L. monocytogenes strains during investigations of food-borne listeriosis.

Two food isolates of Listeria monocytogenes (strains ATCC 51414 and F5027) were sublethally injured by exposure to heat (56°C for 20 min) or to a chlorine sanitizer (Antibac, 100 ppm for 2 min). Percent injury following treatment ranged from 84% to 99%. Injured Listeria were repaired in Listeria repair broth (LRB) at 37°C. Comparison of the repair curves generated by each method indicated that the time for repair was greater for sanitizer-injured cells (14 h) than for heat-injured cells (5 h). Sites of injury were determined by repairing heat- and sanitizer-treated Listeria in LRB supplemented with one of the following inhibitors: rifampicin (10 and 20 μg/ml), chloramphenicol (5 μg/ml), cycloserine D (10 and 20 μg/ml), and carbonyl cyanide m-chlorophenyl-hydrazone(CCCP) (2.5 μg/ml). In both heat- and sanitizer-injured populations, a total inhibition of repair was seen following incubation with rifampicin, chloramphenicol and CCCP. These results clearly indicate a requirement for mRNA, protein synthesis, and oxidative phosphorylation for repair to occur. The cell wall is not a site of damage since cycloserine D had no effect on repair of heat- or sanitizer-injured Listeria. Investigation of damage to the cell membrane showed that stress caused by sublethal heat or sanitizer did not allow proteins or nucleotides to leak into the medium. The recognition of injury and repair in Listeria will lead to improved methods of detection and ultimately to control strategies which prevent outgrowth of this organism in foods.

The cytotoxic effect of culture filtrates from healthy, heat-stressed, and repaired Listeria monocytogenes and L. innocua on the bovine mammary epithelial cell line MAC-T was examined. Culture filtrates were collected from Listeria spp. following treatments which included: (i) 18 h of growth of Listeria at 37°C; (ii) sublethal heat treatment at 56°C for 50 minutes; (iii) repair of the injured cells at 37°C for 7 h; (iv) growth of repaired bacterial cells at 37°C for 36 h; and (v) heat injury at 56°C for 50 min of the cell population obtained after the initial repair and growth. Strains chosen for study included two genetic mutants of L. monocytogenes: a hemolysin-negative mutant, CNL 85/162 (Hly−) and a hemolysin-positive revertant, CNL 85/163 (Hly+). Culture filtrates obtained from Hly− bacteria did not prevent adhesion of the mammary epithelial cells and slightly stimulated their growth. In contrast, culture filtrates from Hly+ bacteria grown for 18 h significantly reduced the ability of MAC-T cells to adhere to the cell culture dishes, prevented the growth of those cells that were attached to the dishes, and caused cell death. Supernatants from Hly+ and Hly− following injury and during repair had no lethal effect on MAC-T cells. The effects of culture medium obtained after growth of the repaired Listeria cells on MAC-T cells were similar to those recorded for medium from the first 18 h growth for both strains, indicating that cells regain virulence potential once they have repaired and reinitiated growth. Culture filtrates obtained from L. monocytogenes Scott A showed results similar to those of Hly+, decreasing adherence and growth of MAC-T cells, while L. innocua culture filtrate had no adverse effect. The results of these experiments suggest that when injured, L. monocytogenes does not demonstrate adverse effects towards MAC-T cells. Once repair is completed and the listeria are growing, activity towards MAC-T cells is restored.