Differential biofilm formation and chemical disinfection resistance of sessile cells of Listeria monocytogenes strains under monospecies and dual-species (with Salmonella enterica) conditions

Abstract

This study aimed to investigate the possible influence of bacterial intra- and interspecies interactions on the ability of Listeria monocytogenes and Salmonella enterica to develop mixed-culture biofilms on an abiotic substratum, as well as on the subsequent resistance of sessile cells to chemical disinfection. Initially, three strains from each species were selected and left to attach and form biofilms on stainless steel (SS) coupons incubated at 15°C for 144 h, in periodically renewable tryptone soy broth (TSB), under either monoculture or mixed-culture (mono-/dual-species) conditions. Following biofilm formation, mixed-culture sessile communities were subjected to 6-min disinfection treatments with (i) benzalkonium chloride (50 ppm), (ii) sodium hypochlorite (10 ppm), (iii) peracetic acid (10 ppm), and (iv) a mixture of hydrogen peroxide (5 ppm) and peracetic acid (5 ppm). Results revealed that both species reached similar biofilm counts (ca. 10(5) CFU cm(-2)) and that, in general, interspecies interactions did not have any significant effect either on the biofilm-forming ability (as this was assessed by agar plating enumeration of the mechanically detached biofilm bacteria) or on the antimicrobial resistance of each individual species. Interestingly, pulsed-field gel electrophoresis (PFGE) analysis clearly showed that the three L. monocytogenes strains did not contribute at the same level either to the formation of mixed-culture sessile communities (mono-/dual species) or to their antimicrobial recalcitrance. Additionally, the simultaneous existence inside the biofilm structure of S. enterica cells seemed to influence the occurrence and resistance pattern of L. monocytogenes strains. In sum, this study highlights the impact of microbial interactions taking place inside a mixed-culture sessile community on both its population dynamics and disinfection resistance.

Fig 1

Biofilm formation (A₅₇₅) on PS microplates by the 11 L. monocytogenes strains (A) and the 8 S. enterica strains (B). Biofilm cells were indirectly quantified by crystal violet staining and absorbance measurements at 575 nm. Monoculture biofilms were left to be formed on PS microplates for 48 h under four different environmental conditions: at 15°C in TSB (□), at 15°C in dTSB (■), at 30°C in TSB (▧), and at 30°C in dTSB (▤). In all cases, growth medium was renewed at 24 h of incubation. The bars represent the mean values ± standard deviations (n = 8, two independent experiments, each performed four times). For each figure separately, mean values sharing at least one common lowercase letter shown above the bars are not significantly different at a P value of <0.05. Strains selected for biofilm formation on SS coupons are included in dashed rectangular boxes.

Fig 2

Biofilm formation (log CFU cm⁻²) on SS coupons by L. monocytogenes (FMCC_B-125, FMCC_B-129, and FMCC_B-169) and S. enterica (FMCC_B-42, FMCC_B-56, and FMCC_B-62) strains. Biofilm cells were quantified by enumeration following detachment and agar plating. Biofilms were left to be formed on SS coupons incubated for 144 h in TSB at 15°C (with medium renewal every 48 h), under three different conditions: (i) monoculture (each strain individually), (ii) monospecies mixed culture (three strains of each one species together), and (ii) dual-species mixed culture (six strains of the two different species together). The bars represent the mean values ± standard deviations (n = 6, two independent experiments, each performed three times). Mean values sharing at least one common lowercase letter shown above the bars are not significantly different at a P value of <0.05.

Fig 3

Log reductions (log CFU cm⁻²) of L. monocytogenes and S. enterica biofilm cells following a 6-min exposure to disinfectants (BC, 50 ppm; NaClO, 10 ppm; PA, 10 ppm; HP-PA, 5 ppm each). Mixed culture biofilms were initially left to be formed on SS coupons incubated for 144 h in TSB at 15°C (with medium renewal every 48 h), either under monospecies or dual-species conditions (three strains per species). The bars represent the mean values ± standard deviations (n = 6, two independent experiments, each performed three times). Mean values sharing at least one common lower-case letter shown above the bars are not significantly different at a P value of < 0.05.

Fig 4

Percentages of L. monocytogenes (open bars) and S. enterica (filled bars) biofilm cells in the dual-species sessile community remaining viable (A) and killed (B) following a 6-min exposure to the following disinfectants: BC at 50 ppm, NaClO at 10 ppm, PA at 10 ppm, and HP-PA at 5 ppm each. Control treatment is without disinfection. Dual-species biofilms were initially left to be formed on SS coupons incubated for 144 h in TSB at 15°C (with medium renewal every 48 h).

Fig 5

Distribution of L. monocytogenes strains (FMCC_B-129, □; FMCC_B-169, ■; and FMCC_B-125, ▧) in mono- and dual-species mixed-culture biofilm communities remaining viable following a 6-min exposure to disinfectants (BC, 50 ppm; NaClO, 10 ppm; PA, 10 ppm; HP-PA, 5 ppm each). Control treatment is without disinfection. Mixed-culture biofilms were initially left to be formed on SS coupons incubated for 144 h in TSB at 15°C (with medium renewal every 48 h).