Survival and recovery of viable but nonculturable Listeria monocytogenes cells in a nutritionally depleted medium.

Survival of a desiccated five-strain Listeria monocytogenes mixture during storage in sand at 4 degrees C for 2 months was determined using the acridine orange direct count method with novobiocin and plate counts. Samples of inoculated sand were taken every 2 weeks, incubated at 37 degrees C for 6 h, stained with acridine orange, and then examined with a fluorescence microscope. Elongated viable but nonculturable cells were most frequently observed during weeks 2 and 4. At weeks 6 and 8, most of the cells either remained viable or were dead. In each microscopic field, only one or two viable but nonculturable cells were observed among hundreds of other viable culturable cells, indicating that L. monocytogenes does not generally become viable but nonculturable. Therefore, viable but nonculturable cells are not a concern when plating environmental samples or desiccated L. monocytogenes cells on nonselective media. Tryptic soy agar with 0.6% (wt/vol) yeast extract (TSAYE) and Columbia agar were used as nonselective plate count media. Modified Oxford agar and TSAYE + 5% (wt/vol) sodium chloride were used as the selective plate count media. The effects of aerobic or anaerobic incubation and media supplementation with 0.1% or 1% (wt/vol) sodium pyruvate were tested to optimize recovery of desiccated cells. Nonselective media showed better recovery when TSAYE and Columbia agar contained 0.1% (wt/vol) pyruvate and were incubated aerobically. These two culture methods were equally effective (P > 0.05) for recovering desiccated L. monocytogenes cells.

Attachment of Listeria monocytogenes on ready-to-eat meats.

Five individual strains of Listeria monocytogenes and a mixed cocktail of all five were studied for attachment on frankfurters, ham, bologna, and roast beef relative to their cell surface characteristics. The ratio of strongly attached (sessile) L. monocytogenes cells compared with total (sessile and planktonic) attached cells on ready-to-eat meats was also determined. Because bacterial cell surfaces were characterized by net negative charge and hydrophobicity, electrostatic interaction chromatography and cationized ferritin methods were chosen to study net negative charge distribution on the bacterial cell surface, whereas hydrophobic interaction chromatography and contact angle measurement were used to examine the cell surface hydrophobicity. No differences (P > 0.05) were observed in cell surface charge or cell surface hydrophobicity among strains. Approximately 84 to 87% L. monocytogenes were found to attach strongly to ready-to-eat meats within 5 min. No differences (P > 0.05) were found among strains or among meats. Micrographs observed from scanning electron microscopy showed no differences among the strains but showed a difference in age of cells (mixed culture) in terms of surface negative charge distribution. More surface negatively charged sites were observed at 0 and 7 days and much fewer at 3 days during storage of washed, harvested cells in buffer at 4 degrees C (aged cells under cold and nutrient deprivation), indicating a possible change in cell surface properties. Because no difference in strains was observed, the contact angle measurement study was carried out with the five-strain mixed culture. The surface hydrophobicity increased in frankfurters, decreased in roast beef, and was unchanged in ham and bologna as a result of inoculation.

Reduction and survival of Listeria monocytogenes in ready-to-eat meats after irradiation.

A five-strain Listeria monocytogenes culture was inoculated onto six different types of ready-to-eat (RTE) meats (frankfurters, ham, roast beef, bologna, smoked turkey with lactate, and smoked turkey without lactate). The meats were vacuum packed and stored at 4 degrees C for 24 h prior to irradiation. Populations of L. monocytogenes were recovered by surface plating on nonselective and selective media. The margins of safety studied include 3-log (3D) and 5-log (5D) reduction of pathogenic bacteria to achieve an optimal level of reduction while retaining organoleptic qualities of the meats. A 3-log reduction of L. monocytogenes was obtained at 1.5 kGy when nonselective plating medium was used. The dosages for 3-log reduction were 1.5 kGy for bologna, roast beef, and both types of turkey and 2.0 kGy for frankfurters and ham on the basis of use of selective medium. The D10-values ranged from 0.42 to 0.44 kGy. A 5-log reduction of L. monocytogenes was obtained at 2.5 kGy with nonselective medium. With selective medium, the dosages were 2.5 kGy for bologna, roast beef, and both types of turkey and 3.0 kGy for frankfurters and ham. Survival of L. monocytogenes in the same RTE meat types after irradiation was also studied. Meats were inoculated with 5 log L. monocytogenes per g and irradiated at doses of 2.0 and 4.0 kGy. Recovery of the surviving organisms was observed during storage at temperatures of 4 and 10 degrees C for 12 weeks. Preliminary results showed no growth in meats irradiated at 4.0 kGy. Survivors were observed for irradiated meats at 2.0 kGy stored at 10 degrees C after the second week. No growth was observed in samples irradiated at 2.0 kGy stored at 4 degrees C until the fifth week.

Survival and recovery of Listeria monocytogenes on ready-to-eat meats inoculated with a desiccated and nutritionally depleted dustlike vector.

Dust from construction was theorized to serve as a vector for L. monocytogenes transmission to ready-to-eat (RTE) meats after heat processing but before packaging. A five-strain Listeria monocytogenes culture including serotype 4b was continually stressed on a sand vector under four sets of nutritionally depleted and dry conditions to simulate postprocessing contamination by dustlike particulates. The stresses included that associated with sand stored at different temperatures (10 and 22 degrees C) and levels of humidity (40% relative humidity [RH], 88% RH, or complete desiccation). Irradiated RTE meats, including frankfurters, bologna, chopped ham, and deli-style roast beef, were inoculated with the L. monocytogenes-contaminated sand every 2 to 3 days over a period of 1 1/2 months. After inoculation, the RTE meats were vacuum packed and stored at 4 degrees C for 24 h. Populations of L. monocytogenes were enumerated by surface plating on nonselective and selective media to recover cells on the basis of the different stresses presented (osmotic or antibiotic). L. monocytogenes was demonstrated to be capable of surviving on the sand vector for > 151 days at 10 degrees C and 88% RH, 136 days at 10 degrees C and 0% RH, 73 days at 22 degrees C and 40% RH, and 82 days at 22 degrees C and 0% RH. These results show that under the most conservative scenario, the 73-day-old L. monocytogenes-contaminated sand was able to attach to and be recovered from the RTE meats. This study illustrated that dust contaminated with L. monocytogenes, once in contact with meat surfaces, can survive and grow, posing a health hazard to consumers.