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

Abstract

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.

FIG. 1.

Example of growth curve of the number of adhering cells of a P. fluorescens population incubated at 10°C in the presence of meat exudate. The inoculum was 10³ CFU·ml⁻¹. The ▴ symbols represent the experimental data. The curve has been fitted to the experimental data using the Microfit software package.

FIG. 2.

Accumulation of CFU (a) and total cells stained by DAPI (b) of P. fluorescens on ceramic tile coupons. The coupons were incubated at 10°C in the presence of meat exudate and subjected to daily cleaning and disinfection (5 min of treatment with Galorox JH at a concentration of 1% and then 5 min of treatment with Galox Azur at a concentration of 0.5%). Meat exudate was deposited onto the coupons after each chemical treatment. The inoculum deposited onto the coupons on d₀ was 10⁵ CFU/ml. Only the cells counted before the daily chemical treatment are shown here. The detection threshold for CFU was −0.4 log(CFU·cm⁻²).

FIG. 3.

Epifluorescence microscopic observation of a 1/10 dilution of the filtrate of the liquid recovering what was detached from a coupon by swabbing and marked with DAPI. (a) Filtrate obtained after cleaning and disinfection on the 10th day of experiment 6 (Table 3). Two cells that appear to have divided are circled. (b) Filtrate obtained after cleaning and disinfection on the eighth day of an experiment conducted without bacterial seeding, with all other conditions being identical to those of the experiments shown in Fig. 2.

FIG. 4.

Epifluorescence microscopic observation of DAPI-stained adhering P. fluorescens cells on randomly chosen places of a same ceramic coupon that had been subjected to repeated chemical treatments (Galorox JH and Galox Azur) and soiling with meat exudate for 16 days. The photographs were obtained on the 17th day before the daily chemical treatment.