Virulence of Culture Filtrate from Heat-Injured and Repaired Listeria Strains: Assay on Bovine Mammary Epithelial (MAC-T) Cells

Abstract

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.

Alkaline phosphatase release assay to determine cytotoxicity for Listeria species

“…Cytotoxicity assays conducted by previous researchers did not include many L. ivanovii strains in their test panel (Del Corral et al 1990 ;Bunduki et al 1996 ;Dallas et al 1996). In this study, it was found that six strains of L. ivanovii had some cytotoxic effect (as indicated by AP release) on Ped-2E9 cells and five strains had a significantly lower cytotoxic effect than L. monocytogenes strains (Fig.…”

“…Rapid detection as well as pathogenicity testing of this organism is essential for controlling incidence of listeriosis. Various mammalian cell lines have been tested for their efficacy to determine virulence of L. monocytogenes in vitro, including intestinal cells (Del Corral et al 1990 ;Pine et al 1991 ;Schlech et al 1994 ;Bunduki et al 1996), fibroblasts (Siddique 1969), and macrophage cells (Portnoy et al 1988 ;Dallas et al 1996). In these cell lines, cytotoxicity was determined by the invasion assay or the trypan blue exclusion test, or by observing morphological changes.…”

Atomic force microscopy investigations of heterogeneities in the adhesion energies measured between pathogenic and non-pathogenicListeriaspecies and silicon nitride as they correlate to virulence and adherence

“…Both soil and glass are substrates to which pathogenic and non-pathogenic Listeria strains frequently attach to in nature(Astwood and Wais 1998; Allan, Yan et al 2004) and during food processing(Thomas, King et al 1991; Hutter and Bechhoefer 1993). The force constant of each cantilever was determined prior to force measurements from the power spectral density of the thermal noise fluctuations in DI water(Bunduki, Zavizion et al 1996; Ayebah, Hung et al 2005). Force measurements were performed under water since water is considered as the main solvent in many in vitro environments such as those used in food-related industries where Listeria cells contaminate inert surfaces(Karunasagar, Krohne et al 1993; Lahellec, Salvat et al 1996; Engelbrecht, Dominguez-Bernal et al 1998).…”

Viability of sublethally injured bacteria of fresh and fresh-cut vegetables from the field through distribution

“…Since E. coli has a teichoic acidlike O-polysaccharide in its surface structure in spite of being a gram-negative bacterium (Perepelov et al 2006) , chlorine stress and fungicide stress may have been more effective in sublethally injuring E. coli and E. coli able environment. In addition, many injured pathogens either retain or exhibit enhanced virulence after resuscitation (McCarthy 1991;Hernández et al 1993;Bunduki et al 1996;Rowe and Kirk 2000) . Therefore, the rejuvenation of injured bacteria can increase the risk of underestimating the microbiological quality and safety of fresh produce from the farm to the table.…”