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The gram-positive bacterium Listeria monocytogenes is a food-borne pathogen with the ability to grow at low temperature. Given the importance of refrigeration as a means of food preservation, the psychrotolerant nature of this microorganism poses a significant public health hazard. In order to better understand the mechanisms underlying cold adaptation of L. monocytogenes, a library of Tn917-lac insertional mutants was screened. A cold-sensitive mutant, named cs1, was isolated and found to be also sensitive to salt-stress. Analysis of the transposon insertion site allowed the identification of a gene, lmo1078, encoding a putative UDP-glucose pyrophosphorylase with 68% identity to GtaB from Bacillus subtilis. In gram-positive bacteria, this enzyme catalyses the formation of UDP-glucose, a precursor of membrane glycolipids and cell envelope teichoic acids. Complementation of mutant cs1 with a wild-type copy of lmo1078 restored its ability to grow at low temperature and high salt concentration, indicating that UDP-glucose pyrophosphorylase activity is important for cold and salt tolerance. These results are thus consistent with previous studies showing the importance of the cell envelope in L. monocytogenes adaptation to stressful conditions.

Cleaning and disinfection of open surfaces in food industry premises leave some microorganisms behind; these microorganisms build up a resident flora on the surfaces. Our goal was to explore the phenomena involved in the establishment of this biofilm. Ceramic coupons were contaminated, once only, with Pseudomonas fluorescens suspended in meat exudate incubated at 10°C. The mean adhering population after 1 day was 10 2 CFU ⅐ cm ؊2 and 10 3 total cells ⅐ cm ؊2, i.e., the total number of cells stained by DAPI (4,6-diamidino-2-phenylindole). The coupons were subjected daily to a cleaning product, a disinfectant, and a further soiling with exudate. The result was a striking difference between the numbers of CFU, which reached 10 4 CFU ⅐ cm ؊2, and the numbers of total cells, which reached 2 ؋ 10 6 cells ⅐ cm ؊2 in 10 days. By using hypotheses all leading to an overestimation of the number of dead cells, we showed that the quantity of nonculturable cells (DAPI-positive cells minus CFU) observed cannot be accounted for as an accumulation of dead cells. Some nonculturable cells are therefore dividing on the surface, although cell division is unable to continue to the stage of macrocolony formation on agar. The same phenomenon was observed when only a chlorinated alkaline product was used and the number of cells capable of reducing 5-cyano-2,3-ditolyl tetrazolium chloride was close to the number of total cells, confirming that most nonculturable cells are viable but nonculturable. Furthermore, the daily shock applied to the cells does not prompt them to enter a new lag phase. Since a single application of microorganisms is sufficient to produce this accumulation of cells, it appears that the phenomenon is inevitable on open surfaces in food industry premises.