Dual-Species Model Biofilm Consisting of Listeria monocytogenes and Salmonella Typhimurium: Development and Inactivation With Cold Atmospheric Plasma (CAP)

Abstract

Most environmental biofilms contain a variety of species. These species can establish cooperative and competitive interactions, possibly resulting in an increase or a decrease in antimicrobial resistance. Therefore, results obtained following inactivation of single-species biofilms by means of different technologies (e.g., Cold Atmospheric Plasma, CAP) should be validated for multi-species biofilms. First, a strongly adherent and mature Listeria monocytogenes and S. Typhimurium dual-species biofilm was developed by altering different incubation conditions, i.e., growth medium, incubation temperature, inoculum ratio of L. monocytogenes and S. Typhimurium cells, and incubation time. Adherence and maturity were quantified by means of optical density measurements and viable plate counts, respectively. Secondly, both the (1 day old) reference biofilm and a more mature 7 days old biofilm were treated for different CAP treatment times (0-30 min). Viable plate counts were again used to determine the (remaining) cell density. For both the biofilm development and inactivation, predictive models were applied to describe the growth/inactivation kinetics. Finally, the kinetics of the [1 and 7 day(s) old] dual-species biofilms were compared with those obtained for the corresponding single-species biofilms. Results implied that a strongly adherent and mature reference dual-species biofilm was obtained following 24 h of incubation at 25°C using 20-fold diluted TSB and an inoculum ratio of 1:1. Main observations regarding CAP inactivation were: (i) the dual-species biofilm age had no influence on the CAP efficacy, although a longer treatment time was required for the oldest biofilm, (ii) for the 1 day old biofilms, CAP treatment became less efficient for S. Typhimurium inactivation when this species was part of the dual-species biofilm, while L. monocytogenes inactivation was not influenced by the biofilm type, and (iii) for the 7 days old biofilms, CAP inactivation of both species became more efficient when they were part of the dual-species biofilms. It can be concluded that the efficacy of the CAP treatment is altered when cells become part of a dual-species biofilm, which is quite important with respect to a possible application of CAP for biofilm inactivation within the food industry.

Keywords: L. monocytogenes; S. Typhimurium; biofilms; cold atmospheric plasma (CAP); dual-species; inactivation; single-species.

Figure 1

Influence of the incubation conditions (i.e., growth medium, incubation temperature, and the ratio of L. monocytogenes and S. Typhimurium in the inoculum) on the adherence (OD) of the dual-species biofilm (n = 3). Biofilms were incubated for 24 h prior to the quantification procedure and were considered to be strongly adherent as the average OD values were higher than their corresponding 4 × OD_C-value. For the influence of the growth medium: for each temperature and each ratio, significant differences have been indicated by means of small letters, with “a” bearing the lowest value. For the influence of the incubation temperature: for each growth medium and each ratio, significant differences have been indicated by means of capital letters, with “A” bearing the lowest value. For the influence of the inoculum ratio: for each growth medium and each incubation temperature, significant differences have been indicated by means of numbers, with “1” bearing the lowest value.

Figure 2

Influence of the incubation conditions (i.e., incubation temperature and the ratio of L. monocytogenes and S. Typhimurium in the inoculum) on the cell density of the dual-species biofilm (n = 3). Biofilms were incubated for 24 h prior to the quantification procedure. For the influence of the incubation temperature: for each ratio, significant differences have been indicated by means of capital letters, with “A” bearing the lowest value. For the influence of the inoculum ratio: for each incubation temperature, significant differences have been indicated by means of small letters, with “a” bearing the lowest value.

Figure 3

Adherence (OD) of the dual-species biofilm as function of the incubation time (n = 5). Biofilms were considered as strongly adherent if the OD of the sample exceeded the 4 × OD_C-value (dash-dot line).

Figure 4

Maturity of the dual-species biofilm as function of time (n = 3). Both the experimental data (symbols) and the global fit (line) of the Baranyi and Roberts (1994) model are represented: total population on general medium (□, black line), L. monocytogenes population on Palcam medium (x, blue line), and S. Typhimurium population on XLD medium (◇, red line).

Figure 5

Cell density [log₁₀ (CFU/cm²)] of the 1 (black) and 7 (red) day(s) old dual-species biofilm as function of the CAP treatment time (n = 3). Both the experimental data (symbols) and the global fit (line) of the Geeraerd et al. (2000) model are represented: total population on general medium (□, solid line), L. monocytogenes population on Palcam medium (x, dashed line), and S. Typhimurium population on XLD medium (♢, dotted line).

Figure 6

Cell density [log₁₀ (CFU/cm²)] of the 1 and 7 day(s) old (i) dual-species biofilm (black), (ii) single-species L. monocytogenes biofilm (red), and (iii) single-species S. Typhimurium biofilm (blue) as function of the CAP treatment time (n = 3). Both the experimental data (symbols) and the global fit (line) of the Geeraerd et al. (2000) model are represented. For the dual-species biofilm: total population on general medium (□, solid line), L. monocytogenes population on Palcam medium (x, dashed line), and S. Typhimurium population on XLD medium (♢, dotted line). For the single-species biofilms: L. monocytogenes population on Palcam medium (x, dashed line) and S. Typhimurium population on XLD medium (x, dotted line).