Development of synthetic media mimicking food soils to study the behaviour of Listeria monocytogenes on stainless steel surfaces.

Listeria monocytogenes is one of the main targets of hygiene procedures in the ready-to-eat food industry due to its ability to persist for months or even years in processing plants, where it can contaminate food during processing. The factors associated with persistence are often those that foster growth, which itself depends on food contamination of surfaces. It is therefore essential to experiment by using food soils or media modelling these soils to understand the behaviour of L. monocytogenes on surfaces of food processing plants. Thus, we set up an experimental plan including three physiological parameters characteristic of the behaviour of cells on surfaces, namely spatial distribution, adhesion forces and the physiological state of sessile L. monocytogenes. These were recorded in two food soils: smoked salmon juice and meat exudate. According to our results, the behaviour of L. monocytogenes on stainless steel surfaces is highly dependent on the food soil used. The presence of viable but non-culturable (VBNC) cells was demonstrated using meat exudate, while all viable cells were recovered using smoked salmon juice. Moreover, on the basis of our criteria and after validation with three strains of L. monocytogenes, we showed that smoked salmon juice can be substituted by a modified culture medium, demonstrating that drawbacks associated with the use of food soils can be overcome.

Impact of cleaning and disinfection on the non-culturable and culturable bacterial loads of food-contact surfaces at a beef processing plant.

We assessed the impact of industrial cleaning and disinfection (C&D) on colony-forming units (CFUs), viable (culturable and viable but non-culturable) cells and on total cells (viable and dead cells). Bacterial loads on polyvinyl chloride (PVC) and stainless steel surfaces in a cutting room at a beef processing plant were determined before and after C&D by real-time PCR to quantify cells from successive swabs from surfaces with or without an ethidium monoazide pre-treatment and by CFU counts on tryptone soy agar. Agar contact plates were also applied after C&D for comparison. Before C&D, total cells reached 5.4 and 4.7 log cells/cm(2), viable cells 4.0 and 4.4 log cells/cm(2) and CFUs 3.1 and 2.9 log CFU/cm(2) on PVC and stainless steel surfaces, respectively. Although C&D left surfaces visually clean, it did not lead to a significant reduction in total cells. Significant reductions were only observed on PVC for CFUs: 0.8 log and on stainless steel surfaces for viable cells and CFUs: 0.8 and 1.5 log, respectively. Our results show that CFUs were both more easily detached and killed on stainless steel surfaces than on PVC surfaces. Other important results include the following observations: 1) a single swabbing detached only between 2 and 27% of the actual bacterial load; 2) after C&D, the difference between the actual culturable population and the one assessed by one agar contact plate was 1.9 and 2.7 log CFU/cm(2) on PVC and stainless steel surfaces, respectively.

Potential of Escherichia coli O157:H7 to persist and form viable but non-culturable cells on a food-contact surface subjected to cycles of soiling and chemical treatment.

Our aim was to assess the potential of Escherichia coli O157:H7 to persist in a processing environment. We studied E. coli behaviour under conditions modelling those of meat plants to establish one initial bacterial load that allows persistence and another that does not. Polyurethane coupons (3.5 cm²) were contaminated once with E. coli in meat exudate before being subjected daily to a cleaning product and a disinfectant, both at half the recommended in-use concentrations, and a further soiling with the exudate. This procedure aimed to model what occurs in harbourage sites. Because previous experiments showed that persistence could not be achieved at 15°C (temperature of slaughter halls), we incubated the coupons at 20°C. Viable cells were determined by ethidium monoazide-qPCR (EMA-qPCR). When the first chemical treatment (CT) was applied to 24-hour biofilms with 5.4 log CFU/cm² cells were no longer detectable after the first week. However, on 66-hour biofilms with 6.7 log CFU/cm², after initially decreasing, E. coli numbers reached 6.6 log CFU/cm² and 8.3 log viable cells/cm² on the 11th day. When E. coli was cultured with a Comamonas testosteroni previously shown to increase E. coli biofilm formation, and subjected to CT on alternate days, E. coli stabilized at 4.6 log CFU/cm² before the CT, from the 5th day of the experiment. The killing and detachment effects of the CT decreased over time and PCR quantification detected a resumption of growth after 2 days (CT on alternate days) or 3 days (daily CT). Intracellular pH (pHi) of individual cells was determined during an experiment in which the CT was applied on alternate days. The proportion of cells with no proton gradient towards the environment (pHi ≤ 5.4) increased after the CT as expected. But during the first week of the experiment only, a further increase in this proportion occurred 24 h after the CT, suggesting that some of the surviving viable but non-culturable cells finally died. This study shows that conditions leading to E. coli O157:H7 persistence are not likely to arise when good refrigeration and hygiene practices are applied, and highlights the usefulness of EMA or PMA-qPCR as a complement to CFU determination in studying bacterial survival after cleaning and disinfection.

The Listeria monocytogenes homolog of the Escherichia coli era gene is involved in adhesion to inert surfaces.

Two transposon-insertional mutants of Listeria monocytogenes showing smaller viable surface-attached cell populations after disinfection with N,N-didecyl-N,N-dimethylammonium chloride were identified. In both mutants, transposon Tn917-lac was found to be inserted into the same gene, lmo1462, which is homologous to the essential Escherichia coli era gene. Both L. monocytogenes lmo1462-disrupted mutants displayed lower growth rates, as was also shown for several E. coli era mutants, and the lmo1462 gene was able to complement the growth defect of an E. coli era mutant. We showed that the disruption of lmo1462 decreased the ability of L. monocytogenes cells to adhere to stainless steel. Our results suggest that this era-like gene is involved in adhesion and contributes to the presence of L. monocytogenes on surfaces.

The lmo1078 gene encoding a putative UDP-glucose pyrophosphorylase is involved in growth of Listeria monocytogenes at low temperature.

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.

First evidence of division and accumulation of viable but nonculturable Pseudomonas fluorescens cells on surfaces subjected to conditions encountered at meat processing premises.

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 degrees C. The mean adhering population after 1 day was 10(2) CFU x cm(-2) and 10(3) total cells x 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 x cm(-2), and the numbers of total cells, which reached 2 x 10(6) cells x 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.

Interactions in biofilms between Listeria monocytogenes and resident microorganisms from food industry premises.

Twenty nine bacterial strains were grown as binary culture biofilms with Listeria monocytogenes to assess their influence on the settlement of the latter on stainless steel coupons. Most of the strains had been isolated from food processing plants after cleaning and disinfection and were tentatively identified by the APILAB Plus 3.3.3 database (bioMerieux). Sixteen of them decreased L. monocytogenes biofilm colony forming units (CFU) counts. Three strains, Bacillus sp. CCL 9 an unidentified Gram-positive strain CCL 59 and Pseudomonas fluorescens E9. 1, led to a 3-log difference in CFU counts between the pure L. monocytogenes biofilms and the mixed biofilms. Eleven strains had no effect and only four, Kocuria varians CCL 73, Staphylococcus capitis CCL 54, Stenotrophomonas maltophilia CCL 47 and Comamonas testosteroni CCL 24, had a positive effect, with a 0.5- to 1.0-log increase in the L. monocytogenes biofilm CFU counts. On its own, L. monocytogenes settled as single cells, but in binary biofilms, different spatial arrangements were observed: (i) with K. varians CCL 73, K. varians CCL 56 and S. capitis CCL 54, L. monocytogenes cells gathered around the microcolonies of the partner strain; (ii) with the two Gram-negative strains, C. testosteroni CCL 24 and CCL 25, L. monocytogenes cells formed its own microcolonies. No link could be found between the exopolysaccharide production capacity of the bacterial strains in pure-culture biofilms and their effect on the L. monocytogenes population in mixed biofilms. With one strain, C. testosteroni CCL 24, adding filter-sterilized supernatant from a pure-culture biofilm to a pure culture of L. monocytogenes increased the number of L. monocytogenes cells adhering to the stainless steel coupons and forming microcolonies. This study suggests that the "house flora" can have a strong effect on the likelihood of finding L. monocytogenes on inert surfaces.